Expression of osteopontin in chronic rhinosinusitis with
血管内皮生长因子在急性肺损伤和急性呼吸窘迫综合征中的研究进展
•424•中国小儿急救医学 2021年 5 月第 28卷第 5期C h i n Pediatr E m e r g M e d，M a y 2021，Vol. 28，N o.5•综述•血管内皮生长因子在急性肺损伤和急性呼吸窘迫综合征中的研究进展彭晓婷K2’3李秋平“2’31南方医科大学第二临床医学院中国人民解放军总医院儿科医学部，北京 100700;2中国人民解放军总医院第七医学中心附属八一儿童医院，北京丨00700;3出生缺陷防控关键技术国家工程实验室，北京100700通信作者：李秋平，Email:zhjhospital@【摘要】急性肺损伤（acute lung injury，A LI)/急性呼吸窘迫综合征（acute respiratory distresssyndrome, ARDS)是临床常见的危重症之一，以失控性的炎症反应、肺泡-毛细血管屏障破坏以及肺泡、肺间质弥漫性水肿为主要病理特征，严重病例可遗留肺纤维化。
基于其病理改变可知，修复受损的肺泡-毛细血管膜以及减轻损伤后的组织纤维化改变是治疗ALI/ARDS的关键。
已知血管内皮生长因子(vascular endothelial growth factor,VEGF)是内皮细胞增殖和分化的主要调控因子，以往研究提示VEGF在ALI/ARDS的炎症调控、组织病理改变中发挥着重要作用，但其扮演的角色极其复杂。
本文主要对VEGF在ALI/A RDS中的表达变化规律、利弊以及调控VEGF信号的效果与机制进行综述。
【关键词】血管内皮生长因子；急性肺损伤；急性呼吸窘迫综合征；表达；调控基金项目：全军医学科技青年培育计划拔尖项目（18QNP007)DOI ： 10. 3760/cma. j. issn. 1673 -4912. 2021.05.018Vascular endothelial growth factor in acute lung iujury and acute respiratory distress syndromePeng Xiaoting1’2,3，Li Qiuping1，2 ,31 Department of Pediatrics, Chinese PLA General Hospital, The Second School o f Clinical Medicine, SouthernMedical University .Beijing 100700，China ;2 Bayi Children's Hospital，Seventh Medical Center o f ChinesePLA General Hospital，Beijing W0700，China./National Engineering Laboratory for Birth Defects Preventionand Control o f Key Technology，Beijing 100700，ChinaCorresponding author：Li Qiuping, Email：zhjhospital@ 163. com【Abstract】Acute lung injury ( ALI)/acute respiratory distress syndrome ( ARDS) is one of the mostcommon clinical critical illnesses in ICU. The basic pathological features of this disease are uncontrolled inflammation, destruction of the alveolar-capillary barrier and diffuse alveolar and interstitial edema. In severecases, patients may develop significant pulmonary fibrosis. Based on the pathological changes, repairing damaged alveolar-capillary membrane and reducing fibrosis seem to be the key to the treatments of ALI/ARDS.Vascular endothelial growth factor( VEGF) is known to be the main regulator of endothelial cell proliferationand differentiation. Previous studies have suggested that VEGF plays an important role in the inflammatoryreactions and pathological manifestations of ALI/ARDS, but the results are complex. This review mainlyfcxused on the expression changes of VEGF in ALI/ ARDS and the effects and mechanisms of regulatingVEGF signaling.【Key words】Vascular endothelial growth factor; Acute lung injury; Acute respiratory distress syn-drome；Expression；RegulationFund program：Youth Training Project for Medical Science and Technology of the PLA( 18QNP007)DOI: 10. 3760/cma. j. issn. 1673-4912. 2021.05.018急性肺损伤（acute lung injury,A LI)/急性呼 吸窘迫综合征（acute respiratory distress syndrome, ARDS)是由重症肺炎、严重感染、休克、大量输血、创伤等非心源性的肺内外致病因素导致的急性、进行性呼吸衰竭[1]。
骨髓炎英文教科书
Chapter 52 - Acute and Chronic OsteomyelitisAnthony R BerendtCarl W NordenEPIDEMIOLOGYThe character of osteomyelitis changed with the advent of antibiotics, evolving from a disease of high mortality to a disease with high morbidity. Certain trends areapparent. Bone and joint tuberculosis has become less common in the developed world, although the advent of HIV-related diseasemay bring about a reversal in thattrend. An increasing number of chronic bone infections are now associated with trauma, surgery and joint replacement rather than being secondary to hematogenousspread.The epidemiology of acute hematogenous osteomyelitis has been detailed.[1]The incidence is higher in males, it varies among geographic areas (Fig. 52.1) and, insome areas, classical acute hematogenous infection is in long-term decline.[2]The male-to-female ratio increases with age from 1.25 in the 0- to 4-year age group to3.69 in the 13- to 19-year age group. There are substantially higher rates in Maori children from New Zealand and Aboriginal children from Western Australia comparedboth with white children living in the same areas and with children living in Europe. Although almost certainly socioeconomic in origin, these differences may also beinfluenced by host genetic factors.There is less clear information on the epidemiology of chronic osteomyelitis, with the exception of diabetic foot infections.[3]There are an estimated 11 million people inthe USA with diabetes; the majority of these have type 2 disease and hence are older adults. Some 3% of diabetic people developa foot ulcer annually and 10–30% ofpatients with an ulcer will eventually need an amputation. Of all amputations in people with diabetes, 60% are preceded by an infected ulcer. Foot problems have beenestimated to be responsible for 15% of the hospital admissions and 25% of the hospital bed usage among diabetic patients. The annual hospital costs for limbamputations that are related to diabetes amount to more than US$350 million. PATHOGENESIS AND PATHOLOGYMicrobial factorsAdhesion is the initial event in the localization of infection.[4]The initial loose adhesion to bone is potentially reversible. However, if the solid phase offers a configuration that is acceptable to the receptors of the micro-organisms, a more permanent adhesion occurs. Staphylococcus aureusstrains possess receptors forextracellular matrix components such as collagen, fibronectin, bone sialoprotein and osteopontin.[5][6]It is unclear which are crucial for the genesis of osteomyelitis, withconflicting data on the role of the collagen receptor. The fibronectin-binding proteins appear to play a role in attachment to,and invasion of, endothelial cells, eventsthat may be of relevance in the earliest stages of hematogenous seeding.[7]It is possible that trauma or injury may expose binding sites for strains of S. aureus.Following adhesion, firm attachment and adherent growth occurs. For staphylococci and some Gram-negative organisms, synthesis of an exocellular polysaccharide(glycocalyx) produces a 'biofilm', within which bacteria can form microcolonies (Fig. 52.2and Fig.52.3). Adherent growth confers phenotypic resistance to antibiotics,probably as a result of changes in cellular metabolism, and the glycocalyx may confer protection against phagocytes and complement.[8]Prostaglandins are potent bone resorption agents that enhance osteoclast activity and collagen synthesis. It was noted in studies of human bone, as well as in studiesof experimental osteomyelitis in animals, that increased production of prostaglandin E2 (the most potent prostenoid in the resorption of bone) was present.[9]Cytokines,in particular tumor necrosis factor, are also potent stimulators of osteoclast action. Finally, it has been recognized that molecules released from a number of thepathogens that cause osteomyelitis are potent stimulators of bone resorption, via cytokine release from monocytes and by stimulation of osteoclast formation.[10]PathologyAs in most organs, an insult to bone is followed by vascular and cellular responses. However, in bone this process is modified by the rigid nature of the bone, becausethe increased tissue pressure cannot be diffused into soft tissue. With increased intramedullary pressure, sinuses and capillaries are compressed in the marrow,producing infarction. At the infarction edge, there is reactive hyperemia, which is associated with increased osteoclastic activity. This in turn produces loss of bone andlocalized osteoporosis. An inflammatory process begins at the margin of the infarcted area and penetrates through the cortex into the subperiosteal area. Becausethere are few anchoring fibers in the periosteum of infants and children, the periosteum is readilystripped from the bone surface by the increased subperiostealpressure. This can result in disruption of the periosteal blood supply to the cortex, and this leads to cortical bone infarction, bone death and sequestrum formation. (Asequestrum is a macroscopic piece of dead bone that is retained within the overall bone structure.) Stripping from underlying bone is an osteogenic stimulus to theperiosteum, which responds by laying down new living bone. The end result is a shell of new bone around or above a dead segment, a response that preserves themechanical strength of the bone, even though parts of it are now dead.Classification systems for osteomyelitisThe most frequently used classification system is that of Waldvogel et al.[11]In this classification, infections are classified as hematogenous, secondary to a contiguousfocus of infection or related to vascular insufficiency. For chronic long bone osteomyelitis, a second classification system, developed by Cierney and Mader,[12]combines four stages of anatomic disease and three categories of physiologic host (Fig. 52.4). This classification is useful for describing severity and location ofinfection and for planning treatment, and it is amenable to study. The three categories of host are:¦ normal except for osteomyelitis¦ systemic or local compromise, and¦ treatment would be worse than the disease.572Figure 52-1 Acute hematogenous osteomyelitis in preschool children. Data from Gillespie.[1]Figure 52-2 Endosteum of bone showing staphylococci near the endosteal haversian canal.In-vitro incubation of bone chips with Staphylococcus aureusinterrupted at 48hours (scanning electromicrograph). From Norden CW, Gillespie WJ, Nade S. Infections in bones and joints. Blackwell Scientific Publications; 1994, with permission.Figure 52-3 Staphylococci enmeshed in glycocalyx near the haversian osteum.In-vitro incubation of bone chips with Staphylococcus aureusinterrupted at 48 hours (scanning electromicrograph). From Norden CW, Gillespie WJ, Nade S. Infections in bones and joints. Blackwell Scientific Publications; 1994, with permission.Figure 52-4 Anatomic classification of osteomyelitis in adult long bones. Adapted with permission from Mader JT, Calhoun J. Osteomyelitis. In: Mandel G, Bennet J, Dolin R, eds.Infectious diseases. New York: Churchill-Livingstone; 1995:1039–52.Causative agents of osteomyelitisIn acute hematogenous osteomyelitis in children, S. aureusaccounts for more than half of the organisms isolated.[13]The next most frequent group of isolates arestreptococci. In osteomyelitis or osteochondritis due to puncture wounds to the foot, Pseudomonas aeruginosais isolated frequently and is associated with the wearingof sneakers. The organism is found in the sole of the sneaker and is presumably carried into the foot by the puncturing nail.[14] Salmonellaspp., although an infrequentoverall cause, are strongly associated with sickle cell disease. In diabetic patients with foot infections, S. aureus, Staphylococcus epidermidis, enterococci, otherstreptococci and Corynebacteriumspp. are among the most frequent aerobic organisms that are isolated from bone. Anaerobic organisms are also frequently isolated,with Peptostreptococcusspp. being most common. Fungi, mycoplasma, mycobacteria, brucella, treponema, actinomycosis and parasites have also all been associatedwith osteomyelitis.573Pathogenesis of diabetic foot osteomyelitisThe pathogenesis of osteomyelitis in the diabetic foot is an important problem that merits additional consideration. Diabetic patients develop foot ulcers because of acombination of motor, sensory and autonomic neuropathy interacting with changes in the mechanical properties of the soft tissues of the foot. Motor neuropathy causesa high-arched foot with clawing of the toes, and this delivers excessive pressures to the metatarsal heads, the heel and the ends of the toes. Subluxation at the metatarsophalangeal joints not only brings the metatarsal head into a more prominent weight-bearing position but also causes the fibrous metatarsal pads to slip outfrom under the metatarsal heads. Sensory neuropathy reduces the response to pain, so that foreign bodies in the shoes are neglected, and clouds the recognition thatit is time to change an ill-fitting pair of shoes or rest the feet. Autonomic neuropathy is associated with excessive fissuringand cracking from dry, poorly lubricated skin,an ideal portal of entry. Nonenzymatic glycosylation of collagen leads to cross-linking, with increased stiffness.These factors together can readily lead to ulceration, which may lead down to a joint or bone. Loss of periosteum causes death of the superficial part of the cortex ofthe bone. Infection can track through the cortical bone into the medulla and spread rapidly up inside the long axis of the bone. Bone infarction and reaction to infectionthen proceeds just as in larger bones. Ischemia from peripheral vascular disease compounds the problem, as may diminished phagocyte function from poor glycemiccontrol.PREVENTIONThere is no known effective method of preventing the development of acute hematogenous osteomyelitis. There is also no proven effective means of preventing thedevelopment of osteomyelitis secondary to bacterial seeding from an infected focus, such as an intravenous catheter. Even with rapid removal of the catheter andtreatment for up to 6 weeks with an antimicrobial agent that is effective against the organism producing the bacteremia, osteomyelitis at a remote site has still beenshown to develop on occasion.[15]Antibiotic prophylaxis has been used successfully to prevent wound infections following surgery for noncompound hip fractures, and it has also been used successfullyin the placement of total hip and knee prostheses.[16]The end point of these studies has been wound infections, but it is reasonable to presume that a certain number ofpatients who develop wound infections could go on to develop infection of the underlying bone and, therefore, antibiotic prophylaxis may play some role in preventingosteomyelitis. In trauma of long bones, aggressive debridement of contaminated and devitalized tissue, with appropriate stabilization and soft tissue cover, has beenshown to reduce rates of infection.[17]Prevention of diabetic foot osteomyelitis involves prevention of ulceration. Patients should have an annual review of their feet with reference to pulses, protectivesensation, ulcers, callosities, evidence of infection, dermatophytosis and footwear. Those with a history of previous ulceration are at high risk of developing furtherulcers and need more frequent review from a trained podiatrist. Ulceration must be promptly treated with the aim of healing thesoft tissues to prevent the entry of newpathogens.CLINICAL FEATURESAcute hematogenous osteomyelitisEarly signs in children, particularly infants, are failure to move the affected extremity and pain on passive movement. These findings in an infant with an acute febrileillness should lead to suspicion of skeletal infection. Soft tissue changes of swelling, redness and heat occur late in osteomyelitis; if found early in the course of illness,one should suspect cellulitis. In older children, the diagnosis is often easier, but it may still be difficult to distinguish between bone and joint infection. Most radiographsdo not show evidence of infection until at least 10–14 days after the onset, but they may show soft tissue changes.In a large series, about 3% of children developed chronic infection as a complication.[13]However, most of these were associated with failure to treat adequately withantibiotics or with significant delays in treatment. Pathologic fractures are rare. If infection involves the growth plate, abnormal growth, resulting in either a shorter orlonger limb, can occur. In young children, infection can track out of the focus in the metaphysis into the joint, because the joint capsule inserts distal to the growth plate.In general, the outcome of acute osteomyelitis in pediatric patients is good, as long as patients are seen within 7–10 days of the onset of illness and treatment is begunand continued for at least 3 weeks.Subacute hematogenous osteomyelitisSome studies suggest that, in temperate zones at least, an increasing proportion of cases present with longer, more insidious histories of pain of more than 2-weekduration, with minimal functional impairment and without systemic illness. The diagnosis of osteomyelitis is generally made when radiology shows a suspicious lyticlesion in the metaphysis, which biopsy shows to be infective. Cultures are usually negative, but the patient responds well to curettage of the lesion andantistaphylococcal antibiotics. One series of such cases represented 7% of all cases of osteomyelitis seen in the reporting hospital over a 9-year period.[18]Chronic osteomyelitis in long bonesChronic osteomyelitis in long bones usually occurs as a result of trauma; less frequently it occurs as a complication of acute hematogenous osteomyelitis. Patientsusually report few systemic symptoms but are commonly troubled by persistent pain and drainage through sinus tracts. Following successful treatment, many patientscomment on the dramatic improvement in overall physical well-being, and some patients gain weight. The fundamental problem is the prolonged persistence of viablepathogens. The process involves the consequences of continuing necrosis, such as sequestrum and sinus formation, versus repair with new bone formation and scar (Fig 52.5and Fig 52.6).Figure 52-5 Chronic osteomyelitis.The patient is a 30-year-old man who was born in Pakistan and who, as a child, had chronic osteomyelitis caused by Staphylococcus aureus. Heis asymptomatic now except for occasional pain in the hip and a limp. The radiograph shows destruction of the femoral head and acetabulum, chronic changes in the femoral shaft and fusion of theright hip joint. Courtesy of Dr Joseph Mammone.574Figure 52-6 Chronic active osteomyelitis in the femur.This case of osteomyelitis was secondary to a fracture and open reduction and internal fixation 30 years before. Thisaxial, contrast-enhanced, fat-suppressed T1-weighted MRI scan shows cortical thickening and a focal intraosseous fluid collection with an enhancing rim, communicating via a sinus tract to the surfaceof the thigh (arrow).Potential complications of chronic osteomyelitis include septic arthritis if infection tracks into a joint, pathologic fracture, septicemia if a draining sinus becomes blockedand secondary amyloidosis, which is a rare occurrence (one series reported an incidence of about 1%).[19]A second rare complication, long recognized, is thedevelopment of squamous cell carcinoma in scar tissue. Again, the incidence is low (probably less than 1%) and those cases thathave been reported occurred after anaverage of 27 years of osteomyelitis with drainage. The clinical features that are characteristic of malignancy include increased pain, increased drainage, odor and amass. There was usually more radiographic evidence of bone destruction than is seen in patients with uncomplicated osteomyelitis.Vertebral osteomyelitisThe most typical presentation of vertebral osteomyelitis is back pain. The pain is increased by loading the spine and relieved by rest. The degree of pain may seem outof proportion to the examination; it is unusually severe, and night pain is an important feature. In about 10% of patients, symptoms may be present for less than 1 weekandFigure 52-7 Vertebral osteomyelitis.A sagittal, contrast-enhanced convential spin echo MRI scan (T1-weighted) demonstrates a posteriorly located epidural abscess at the L4–L5vertebral level with an enhancing rim and displacement of the nerve roots anteriorly. Courtesy of Dr Joseph Mammone.the illness appears more severe with fever, night sweats and other systemic signs of infection. In such patients, blood cultures are usually positive. The majority have asubacute presentation with symptoms of back pain that are present for anywhere from 2 weeks to 2 years before diagnosis. Generally, only about half of the patientsare febrile on initial evaluation.The major complications of vertebral osteomyelitis are neurologic symptoms, caused by retropulsion of disc material, an inflammatory mass or an associated epiduralabscess.[20]The classic clinical progression goes from spinal ache to root pain to weakness, followed by paralysis. Careful and repeated examination of patients withvertebral osteomyelitis is critical; if such symptoms begin, they should be investigated rapidly with radiologic studies, particularly magnetic resonance imaging (MRI; Fig52.7and Fig 52.8). Urgent surgical decompression is often needed.[21]Unfortunately, the neurologic complications of epidural abscess are not always reversible, so the goal of management should be detection at the earliest stage (Fig. 52.9).Bone infections that underlie pressure soresPatients with osteomyelitis beneath pressure sores are immobile, insensate at the pressure area or malnourished; they may be all of these things. Osteomyelitispresents as a failure of the patient to thrive, and a failure of the wound to heal, despite optimal nursing care and offloadingof the sore.[22]There may be pain, but it isoften not prominent. Systemic illness is ominous, implying the development of septicemia or the formation of an abscess. Depending on the degree of local sensoryimpairment and the location of the sore, underlying collections of pus can be very extensive. So too can be the extent of the wound, which may be deeply underminedand sloughy at presentation.Special patient populationsPatients undergoing hemodialysisIn patients undergoing hemodialysis who present with bony pain or fractures, there must be a high index of suspicion of bone infections. Bone biopsy is necessary tomake the diagnosis and to identify the infecting agent because the clinical signs, radiographic picture and symptoms can mimic those of renal osteodystrophy.[23]Theusual infecting organisms are staphylococci (either S. aureusor S. epidermidis) or P. aeruginosa. Figure 52-8 Vertebral osteomyelitis.A sagittal, turbo spin echo MRI scan (T2-weighted) from the same patient as the scan in Fig. 52.7. Courtesy of Dr Joseph Mammone.575Figure 52-9 Vertebral osteomyelitis.A myelogram showing posterior compression of the spinal cord by an inflammatory mass. Note the involvement of adjacent vertebral endplates andthe intervertebral disc. Courtesy of Dr Joseph Mammone.Intravenous drug usersAlthough septic arthritis is more common than osteomyelitis in intravenous drug users, the diagnosis must be suspected if bone pain is present. Pain and tendernessare common. In general, the organisms isolated from bone are S. aureus, streptococci or P. aeruginosa; P. aeruginosainfection is presumably due to the use ofnonsterile water for injecting drugs.Osteomyelitis in the diabetic footDiabetic patients rarely manifest a fever with foot infections. Systemic illness indicates severe disease and at a local level,there is usually some accompanyingnecrosis, gangrene, fasciitis, severeFigure 52-10 Osteomyelitis in a diabetic patient.Diabetic patient with osteomyelitis and destruction of proximal second phalanx and metatarsal as well as secondmetatarsal-phalangeal joint. Courtesy of Dr Joseph Mammone.cellulitis or significant swelling of the foot indicating a deep abscess. Most patients appear well, although with some worsening of glycemic control. Despite neuropathy,there is often some pain to accompany an ulcer, with signs of soft tissue infection, purulence, erythema, swelling and local warmth. Depending on the depth and extentof the ulceration, bone, cartilage, joint capsule or tendon may be visible in the wound. Callosities indicate chronic excessiveweight-loading on a particular area, and it isoften beneath these that tissue breakdown occurs. Hemorrhage beneath a callosity is often associated with tissue breakdown or infection. Feet of this kind are oftennot properly evaluated during admission of the patient to a general hospital (Fig. 52.10).[24]DIAGNOSISThe diagnosis of osteomyelitis requires clinical suspicion, a consistent history and physical examination, and supportive laboratory studies (both radiographic andmicrobiologic). Certain conditions mimic osteomyelitis, and these differential diagnoses are reviewed briefly below.Acute osteomyelitisThe diagnosis of acute hematogenous osteomyelitis is essentially a clinical one assisted by some of the studies discussed below. In the absence of a clear cause,limping or pain in an extremity should raise the suspicion of infection of bone. The sedimentation rate and C-reactive protein are frequently elevated in the presence ofosteomyelitis (in 96% and 89% of cases, respectively), but normal values do not exclude the diagnosis.[25]Blood cultures are positive in just over 50% of cases. Plainradiographs may show soft tissue swelling but are otherwise usually normal because it takes anywhere from 10 to 14 days to destroy 50% of the bone (which is theamount of destruction required to show up as a lesion on conventional radiography). Ultrasonography has been reported to be successful in detecting subperiosteal abscess in the presence of acute osteomyelitis; deep soft tissue swelling is the earliestsign of acute osteomyelitis, followed by periosteal elevation and a thin layer of periosteal fluid, which, in some cases, progresses to form a subperiosteal abscess.[26][27]These later stages were marked by cortical erosion; this sign generally appears only in patients who have had symptoms for morethan 1 week.Technetium bone scans are exquisitely sensitive and are generally positive before lesions appear on radiograph; however, false-negative bone scans have beenreported when the diagnosis of acute osteomyelitis has been confirmed by aspiration of pus. [28]The ultimate diagnostic test in acute osteomyelitis is growth of the infecting pathogen in cultures of purulent material obtained by needle aspiration from the painfulinfected area. Complex tests such as indium-labelled white blood cell scan, computerized tomography (CT) scanning and MRI have little place in the management ofacute hematogenous osteomyelitis unless for surgical planning or to confirm the diagnosis. In any even, treatment of the patient with suspected acute osteomyelitismust not wait for diagnostic imaging.Chronic osteomyelitisIn contrast to acute osteomyelitis, investigation and diagnosis can generally precede antibiotic therapy. Given a clinical suspicion of chronic osteomyelitis, the clinicianhas a plethora of diagnostic studies to choose from.[28]Unfortunately, none is perfect (Table 52.1). An algorithm is offered for the approach to suspectedosteomyelitisand its management (Fig. 52.11). In a nondiabetic patient, if the plain radiograph is positive for osteomyelitis, it is possible to proceed directly to bonebiopsy fordetermination of the infecting organism and its antimicrobial susceptibility. The features of rapidly progressive,576TABLE 52-1-- Tests for osteomyelitis.*TESTS FOR OSTEOMYELITISSensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%)Three-phase bone scan 95 33 53 90Gallium scan 81 69 71 80Indium-labeled white blood cell scan 88 85 86 87MRI 95 88 93 92Sensitivity, specificity, positive predictive values and negative predictive values of tests used to diagnose infection of bone.* Adapted from White et al.[30]Figure 52-11 Investigation and management of chronic osteomyelitis.mixed destructive and reparative bone responses are highly distinctive. If the radiograph is normal and osteomyelitis is suspected, one may go directly to a three-phasebone scan, a labelled white cell scan or MRI. Indeed, MRI is particularly valuable in that it shows the extent of infection inside the bone, the presence of soft tissueabnormalities, including abscesses and sinus tracts, and erosions or breaches of the cortex. Its very high sensitivity and specificity have made it the imaging modality ofchoice in osteomyelitis,[29]although577its value is reduced in patients who have present or past metal work (because of signal void from implants or metallosis) and in patients with recent surgery, whichcauses marrow edema in its own right.Ultimately, the procedure of choice is bone biopsy, often referred to as the gold standard for osteomyelitis. The test is easily done, but the rate of false-negative resultshave been reported in some series as being as high as 65%, probably because osteomyelitis has a patchy distribution in the bone. All specimens should be sent forboth histology and microbiology. In one well-done study, in which 16 biopsy specimens demonstrated histologic evidence of osteomyelitis, only eight were alsoculture-positive.[30]In the same study, if either histology or culture was considered a positive criterion for osteomyelitis, the positive predictive value was 100% and thenegative predictive value was 66%. Obviously, the larger the amount of bone sampled, the more biopsies taken and the better theimaging guidance, the more likelyone is to get a positive biopsy. Finally, it should be noted that, in diagnosing osteomyelitis, sinus tract cultures have little value and correlate poorly with the organismsfound in specimens taken in the operating room.[31]Therefore, the results of cultures of draining sinuses should not be relied on to identify the causative pathogen.Bone infections underlying pressure soresConfirming the diagnosis of osteomyelitis beneath a pressure sore can be difficult. Radiographic or nuclear imaging and soft tissue cultures can be abnormal in thearea of a pressure sore and may suggest osteomyelitis when none is present. Such misdiagnosis can lead to prolonged and potentially toxic courses of antimicrobialagents.A careful study of bone infections and pressure sores made several valuable points:[22]¦ the diagnosis of underlying bone infection should be considered whenever a pressure sore does not heal;¦ clinical evaluation of the depth of the sore or its duration is not helpful in determining whether bone infection is present;¦ failure of the sore to close after pressure is removed is helpful in determining whether there is underlying osteomyelitis;¦ nuclear scans are generally useful only if negative — the negative predictive value was high;¦ Gram-negative bacilli, anaerobes and streptococci are most often cultured from infected bone; and¦ bone biopsy histology and culture are the gold standard in diagnosing osteomyelitis —the procedure is rarely associated withcomplications. Biopsy must,however, be taken through uninvolved skin if done percutaneously, or after debridement of overlying tissue if at operation, to avoid culturing surface contaminants.Osteomyelitis in the diabetic footIn diabetic patients, the approach to diagnosing osteomyelitis in the foot (the usual site of the disease) is somewhat different. Conventional radiographs may bediagnostic if there is rapid progression of changes (e.g. over 2–3 weeks), but it can be extremely difficult to distinguish diabetic osteopathy from osteomyelitis. Becauseosteopathy will not respond to antimicrobial agents, this distinction is critical. Nuclear medicine scans are often difficult to interpret because there is soft tissue infectionand it is difficult to localize infection to bone as opposed to the soft tissue (Fig. 52.12and Fig.52.13). One of the simplest tests is to take a steel probe and insert itinto the ulcer; contact by the probe with bone has a high correlation with the presence of osteomyelitis.[32]。
中医药治疗胰腺纤维化的临床对策及研究进展
·综述·DOI： 10.3969/j.issn.1001-5256.2023.09.034中医药治疗胰腺纤维化的临床对策及研究进展纪晓丹1，龚彪1，李兴佳1，吕婵1，徐莹21 上海中医药大学附属曙光医院消化科，上海 201203；2 上海中医药大学教学实验中心，上海 201203通信作者：徐莹，******************（ORCID： 0000－0002－4645－3094）摘要：胰腺纤维化是慢性胰腺炎疾病发展不可逆的主要病理变化，目前临床针对胰腺纤维化的治疗仍缺乏疗效确切的药物。
本文总结了近年关于中医药治疗胰腺纤维化的临床策略及研究进展。
中医辨证胰腺纤维化涉及到的脏腑有肝、胆、脾、胃；病理因素与火、瘀血、痰湿相关；中药提取物抗胰腺纤维化的相关研究涉及的药物类别包括健脾类、化湿类及化瘀类等，中药方剂治疗胰腺纤维化的相关机制信号通路主要是干预胰腺星状细胞的激活。
以上研究为中医药对胰腺纤维化的预防、干预及防治并发症的深入探索提供了参考。
关键词：胰腺炎，慢性；纤维化；中医药疗法基金项目：国家自然基金青年科学基金项目（82004162）；上海市青年科技英才扬帆计划（20yf1449500）；上海中医药大学附属曙光医院“四明青年基金”（SGKJ－201924）Application of traditional Chinese medicine in treatment of pancreatic fibrosis：Clinical strategies and research advancesJI Xiaodan1，GONG Biao1，LI Xingjia1，LYU Chan1，XU Ying2.（1. Department of Gastroenterology，Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine，Shanghai 201203，China；2. Teaching and Experiment Center of Shanghai University of Traditional Chinese Medicine， Shanghai 201203， China）Corresponding author： XU Ying，******************（ORCID： 0000－0002－4645－3094）Abstract：Pancreatic fibrosis is the main irreversible pathological change during the progression of chronic pancreatitis， and at present，there is still a lack of effective drugs for the treatment of pancreatic fibrosis in clinical practice. This article summarizes the application of traditional Chinese medicine （TCM） in the treatment of pancreatic fibrosis in recent years from the aspects of clinical strategies and research advances. The TCM syndrome differentiation of pancreatic fibrosis involves the liver，gallbladder，spleen，and stomach，and pathological factors are associated with fire，blood stasis，and phlegm dampness. The research on the anti－pancreatic fibrosis effect of TCM extracts mainly involves spleen－strengthening，dampness－resolving， and blood stasis－resolving drugs， and intervention against the activation of pancreatic stellate cells is the main signaling pathway involved in the mechanism of TCM prescriptions in the treatment of pancreatic fibrosis. The above studies provide a reference for in－depth research on the application of TCM in the prevention and intervention of pancreatic fibrosis and the prevention and treatment of related complications.Key words：Pancreatitis， Chronic； Fibrosis； Traditional Chinese Medicine TherapyResearch funding：National Natural Science Fund for Youth （82004162）； Shanghai Young Science and Technology Talents Sailing Program （20yf1449500）；“Siming Youth Fund” of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine （SGKJ－201924）胰腺纤维化是慢性胰腺炎疾病发展的主要病理变化［1］，在临床上针对慢性胰腺炎的治疗主要以改善疼痛、预防其急性发作，纠正胰腺内外分泌功能不全及防治并发症为主［2］。
火龙罐疗法联合常规治疗在AECOPD_患者中的应用效果
- 143 -*基金项目：广东省中医药局立项项目（202105211511578370）①梅州市中医医院 广东 梅州 510000火龙罐疗法联合常规治疗在AECOPD患者中的应用效果*韩文聪① 陈晓英① 郑小芬① 邹爱萍①【摘要】 目的：探讨火龙罐疗法联合常规治疗在慢性阻塞性肺疾病急性加重期（AECOPD）患者中的应用效果。
方法：选取2022年1—10月在梅州市中医医院住院的100例AECOPD 患者作为研究对象，根据临床实际情况以及患者意愿，且按是否接受火龙罐治疗分为治疗组与对照组，各50例。
对照组给予常规治疗，治疗组给予火龙罐疗法联合常规治疗。
比较两组治疗前后症状积分、血气指标[动脉血氧分压（PaO 2）、动脉血二氧化碳分压（PaCO 2）]，治疗前及随访3个月、6个月生活质量（ADL）与6分钟步行试验（6MWT）变化，不良反应发生情况。
结果：治疗后，两组症状积分、PaCO 2较治疗前降低，且治疗组低于对照组，两组PaO 2较治疗前提高，且治疗组高于对照组，差异有统计学意义（P <0.05）。
随访3个月、6个月，两组ADL 评分、6MWT 距离较治疗前提高，随访6个月两组各指标较随访3个月提高，且治疗组高于对照组，差异有统计学意义（P <0.05）。
两组治疗期间不良反应发生率比较，差异无统计学意义（P >0.05）。
结论：火龙罐疗法联合常规治疗AECOPD 效果满意，可缓解患者的临床症状，改善血气指标，提高生活质量及步行能力，同时不增加不良反应发生率。
【关键词】 慢性阻塞性肺疾病急性加重期 火龙罐疗法 康复 血气指标 doi：10.14033/ki.cfmr.2024.06.036 文献标识码 B 文章编号 1674-6805（2024）06-0143-04 Application Effect of Fire Dragon Cup Therapy Combined with Conventional Treatment in Patients with AECOPD/HAN Wencong, CHEN Xiaoying, ZHENG Xiaofen, ZOU Aiping. //Chinese and Foreign Medical Research, 2024, 22(6): 143-146 [Abstract] Objective: To investigate the application effect of Fire Dragon Cup therapy combined with conventional treatment in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Method: A total of 100 patients with AECOPD who hospitalized in Meizhou Traditional Chinese Medicine Hospital from January to October 2022 were selected as the research objects, according to the actual clinical situation and patients' wishes, they were divided into the treatment group and the control group according to whether they received Fire Dragon Cup treatment, with 50 cases in each group. The control group was given conventional treatment, and the treatment group was given Fire Dragon Cup therapy combined with conventional treatment. The symptom scores, blood gas indexes [arterial partial pressure of oxygen (PaO 2), arterial partial pressure of carbon dioxide (PaCO 2)] before and after treatment, quality of life (ADL) and 6-minute walking test (6 MWT) before treatment, 3 months and 6 months after follow-up, and incidence of adverse reactions were compared between two groups. Result: After treatment, the symptom scores and PaCO 2 of two groups were lower than those before 弹力纤维降解的影响及机制[J].中华实用诊断与治疗杂志，2020，34（1）：9-12.[19] SAN NORBERTO E M，REVILLA Á，Fernandez-Urbon A，et al.Vascular calcification progression in patients with end-stage chronic kidney disease[J]. Int Angiol，2021，40（6）：528-536.[20] LI H，YANG M. Ligustrazine activate the PPAR-γ pathwayand play a protective role in vascular calcification[J]. Vascular，2022，30（6）：1224-1231.[21] WING T T，ERIKSON D W，BURGHARDT R C，et al. OPNbinds alpha V integrin to promote endothelial progenitor cell incorporation into vasculature[J]. Reproduction，2020，159（4）：465-478.[22] OGATA H，FUKAGAWA M，HIRAKATA H，et al. Effect oftreating hyperphosphatemia with lanthanum carbonate vs calcium carbonate on cardiovascular events in patients with chronic kidney disease undergoing hemodialysis: the LANDMARK randomized clinical trial[J]. JAMA，2021，325（19）：1946-1954.[23] SHIRAKAWA K，SANO M. Osteopontin in cardiovasculardiseases[J]. Biomolecules，2021，11（7）：1047.[24] DE VRIESE A S，CALUW É R，PYFFEROEN L，et al.Multicenter randomized controlled trial of Vitamin K antagonist replacement by Rivaroxaban with or without Vitamin K 2 in hemodialysis patients with atrial fibrillation: the Valkyrie Study[J]. J Am Soc Nephrol，2020，31（1）：186.（收稿日期：2023-08-22） 慢性阻塞性肺疾病（COPD）属于常见的一种慢性气道疾病，COPD分为稳定期与急性加重期[1]。
甲状腺癌与乳腺癌关系研究进展
了 在 母 乳 中 作 为 一 种 营 养 素 外 ，其 在 正 常 或 患 病 的 乳房中尚无已知的作用。乳腺癌 组 织 能 够 吸 收 放 射性碘，而正常的、非泌乳的乳腺组织并不摄取[29]。 上述研究为育龄女性易患乳腺癌提供了可能的 依据。 4 甲状腺癌与乳腺癌的临床特征
有 研 究 表 明 ，乳 腺 癌 与 甲 状 腺 癌 存 在 一 定 的 关 系 ，两者中任一种癌症患者发生另一种癌症的风险 均高于正常人群[3°_32]。乳腺癌 女 性 患 非 恶 性 甲 状 腺 结 节 比 例 高 于 无 乳 房 肿 瘤 女 性 [33]。值 得 注 意 的 是 ，甲状腺乳头状癌、甲状腺滤泡癌和乳腺浸润性 导管癌或导管内癌是甲状腺合并乳腺癌患者的主 要 病 理 类 型 。对 甲 状 腺 癌 患 者 或 乳 腺 癌 患 者 治 疗 后随访应该常规行甲状腺和乳腺的相关检查[34]。 5 小结
甲状腺癌合并乳腺癌的发生原因目前仍未十 分 明 确 。有 研 究 表 明 ，乳 腺 癌 和 甲 状 腺 癌 具 有 一 些 共同的致病因素，如 激 素 、肿瘤基因易感性、肥 胖 、机 体免疫力或者免疫监控能力下降、放疗与化疗等[7]。 2. I 激 素 乳 腺 癌 通 常 由 雌 激 素 和 孕 激 素 受 体 上
参考文献：
[ 1 ] Marcheselli R , Marcheselli L.Cortesi L ,et al. Risk of second pri mary malignancy in breast cancer survivors：a nested populationbased case-control s tu d y [ J ] .J Breast C ancer,2 0 1 5 ,1 8 ( 4 ) ：378385.
肾素抑制剂阿利吉仑在非糖尿病肾小球疾病治疗中的应用前景
肾素抑制剂阿利吉仑在非糖尿病肾小球疾病治疗中的应用前景非糖尿病性肾小球疾病是导致终末期肾病（end stage renal disease，ESRD）的常见原因。
慢性肾脏病进展到一定阶段后均通过肾小管间质纤维化共同途径发展到ESRD，该过程中肾素-血管紧张素-醛固酮系统（RAAS）扮演重要的作用。
阻断RAAS系统能延缓慢性肾脏病的进展；ACEI或ARB在肾脏疾病治疗上确实起到重要作用，但仍不令人满意，部分患者蛋白尿仍持续存在。
阿利吉仑是第一个非肽类直接肾素抑制剂，不仅可直接作用于RAAS，还可抑制PRR/MAPK/ERK信号通路而起到抗纤维化作用。
本文致力于阐述其应用于肾小球疾病的治疗前景。
[Abstract]Non-diabetic glomerulonephritis is a common cause of end stage renal disease （ESRD）.The development of chronic kidney diseases （CKD）to a certain stage is through the common pathway of renal tubule interstitial fibrosis to ESRD，and renin-angiotensin-aldosterone system （RAAS）plays an important role during the process.Blocking RAAS system can delay the progress of CKD.Angiotensin-converting enzyme inhibitor （ACEI）or angiotensin receptor blocker （ARB）exerts a great role，but which still is not satisfying due to persisted proteinuria in some patients.Aliskiren is the first non-peptide direct renin inhibitor，which not only directly influences on RAAS，also blocks the PRR/MAPK/ERK signal pathway with anti-fibrosis.The paper focused on the prospect of applying Aliskiren to glomerulopathy.[Key words]Non-diabetic glomerulonephritis；Renin-angiotensin-aldosterone system；Aliskiren腎素-血管紧张素-醛固酮系统（renin-angiotensin-aldosterone system，RAAS）是体内调节血压及血容量的关键系统。
G蛋白偶联雌激素受体对乳腺癌患者预后和临床病理的影响：系统荟萃分析
doi:10.3971/j.issn.1000-8578.2023.23.0237·临床研究·G蛋白偶联雌激素受体对乳腺癌患者预后和临床病理的影响：系统荟萃分析刘龙娇，姚宇锋Effect of G-protein-coupled Estrogen Receptor on Prognosis and Clinicopathological Characteristics of Patients with Breast Cancer: A Systematic Meta-analysisLIU Longjiao, YAO YufengDepartment of General Surgery, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing Medical University Affiliated Cancer Hospital, Nanjing 210000, China CorrespondingAuthor:YAOYufeng,Email:**********************Abstract: Objective In this study, a meta-analysis of the expression of G-protein-coupled estrogen receptor (GPER) in breast cancer (BC) and its role in prognosis was conducted to understand the effect of this expression on the survival and clinicopathological characteristics of patients with BC. Methods Identical search strategies were used to search relevant literature in electronic databases updated to November 24, 2022. Individual hazard ratios (HR s) and odds ratios (OR s) with their 95%CI were extracted and pooled to evaluate the strength of the association between positive GPER expression and survival results, the clinicopathological features of patients with BC. Begg’s tests, Egger’s tests, and funnel plots were used to evaluate publication bias. Heterogeneity and sensitivity were also assessed. All works were completed using Review Manager 5.4.1. Results GPER expression had a favorable effect on OS (HR=0.77; 95%CI: 0.49-1.22; Z=1.10; P=0.27) and an unfavorable effect on DFS/RFS/DDFS (HR=1.03; 95%CI: 0.64-1.65; Z=0.13; P=0.90) in patients with BC. GPER expression was not significantly related to the prognosis of patients with BC, and GPER expression was not an independent prognostic factor. Furthermore, GPER expression was significantly associated with TNM staging (OR=0.31, 95%CI: 0.06-0.55, Z=2.43, P=0.02), distant metastasis (OR=6.82, 95%CI: 1.89-24.55, Z=2.94, P=0.003), histological grade (OR=0.009, 95%CI: −0.16-0.01, Z=2.16, P=0.03), ER expression (OR=1.77, 95%CI: 1.15-2.72, Z=2.59, P=0.009), and PR expression (OR=1.36, 95%CI: 1.00-1.84, Z=1.95, P=0.05). Conclusion GPER may not be an independent prognostic factor for BC. GPER expression was significantly related to some clinicopathological features of patients with BC, including TNM staging, distant metastasis, histological grade, ER expression, and PR expression.Key words: GPER; Breast cancer; System meta-analysis; Prognosis; Clinicopathological characteristicsCompeting interests: The authors declare that they have no competing interests.摘 要：目的 本研究对G蛋白偶联雌激素受体（GPER）在乳腺癌中的表达及其对预后的作用进行荟萃分析，以了解GPER表达对乳腺癌患者生存和临床病理特征的影响。
生物学标志物对慢加急性肝衰竭早期预后的评估
㊃专题㊃通信作者:冯志杰,E m a i l :z h i j i e f e n g2005@163.c o m 生物学标志物对慢加急性肝衰竭早期预后的评估刘慧敏,冯志杰(河北医科大学第二医院消化内科,河北省石家庄050000) 摘 要:慢加急性肝衰竭(a c u t e -o n -c h r o n i c l i v e r f a i l u r e ,A C L F )病情危重㊁进展迅速,预后不佳,早期识别并积极治疗是患者存活的关键㊂近年来国内外涌现出的多种预测A C L F 的新型生物学标志物,经分析汇总归纳为四类:蛋白质类(包括炎症反应㊁细胞凋亡或坏死以及免疫反应相关标志物)㊁细胞类㊁核酸类和代谢产物类,其中部分生物学标志物可能具有良好的预后评估价值㊂关键词:肝功能衰竭;预后;生物学标记中图分类号:R 575.3 文献标志码:A 文章编号:1004-583X (2018)11-0933-05d o i :10.3969/j.i s s n .1004-583X.2018.11.004E a r l yp r o gn o s t i c e v a l u a t i o no f b i o m a r k e r s f o r a c u t e -o n -c h r o n i c l i v e r f a i l u r e L i uH u i m i n ,F e n g Z h i ji e D e p a r t m e n t o f G a s t r o e n t e r o l o g y ,t h eS e c o n d H o s p i t a l o f H e b e iM e d i c a lU n i v e r s i t y ,S h i j i a z h u a n g 050000,C h i n a C o r r e s p o n d i n g a u t h o r :F e n g Z h i j i e ,E m a i l :z h i j i e f e n g2005@163.c o m A B S T R A C T :A c u t e -o n -c h r o n i c l i v e r f a i l u r e i s c h a r a c t e r i z e db y c r i t i c a l c o nd i t i o na n dr a p i dde v e l o p m e n tw i t h p o o r p r o g n o s i s .E a r l y d i a g n o s i s a n d p o s i t i v e t r e a t m e n t i s t h ek e y t os u r v i v a l of p a t i e n t s .N u m e r o u s p r o gn o s t i cb i o m a r k e r s a r i s i n g i n r e c e n t y e a r sc a nb es u mm a r i z e da n dd i v i d e di n t of o u rc a t e g o r i e s ,i n c l u d i n gp r o t e i n s (b i o m a r k e r sc o r r e l a t e w i t hi n f l a mm a t i o n r e s p o n s e ,a p o p t o s i s o r n e c r o c y t o s i s ,a n d i mm u n o r e a c t i o n ),c y t o l o g y ,n u c l e i c a c i d s ,a n d m e t a b o l o m i c s .P a r t o f t h e m m a y h a v eb e t t e r p r o gn o s t i c e v a l u a t i o nv a l u e .K E Y W O R D S :l i v e r f a i l u r e ;p r o gn o s i s ;b i o m a r k e rs 河北医科大学第二医院主任医师㊁教授,硕士生导师,消化内科主任㊂兼任中国医师协会内镜分会消化内镜专业委员会委员㊁中华医学会消化内镜分会超声内镜学组委员㊁中华医学会消化内镜分会食管协作组委员㊁河北省医学会消化内镜学分会主任委员㊁河北省急救医学会消化专业委员会主任委员㊁河北省药学会消化药学专业委员会主任委员㊁河北省医学会消化内镜学会超声内镜学组组长㊁河北省医学会肝病学分会常委等㊂主要研究方向为消化内镜技术的应用,肝硬化门脉高压㊁急性胰腺炎的诊治等㊂先后独立承担京津冀基础研究合作专项项目1项,河北省政府资助专科能力建设项目1项,河北省医学适用技术跟踪项目4项等课题㊂发表论文100余篇,参编著作7部㊂获河北省科技进步三等奖3项,获河北省卫生厅医学成果二等奖1项㊂慢加急性肝衰竭(a c u t e -o n -c h r o n i c l i v e r f a i l u r e ,A C L F )是以慢性肝病出现急性肝功能失代偿㊁器官衰竭㊁高短期死亡率为特征的一种综合征㊂全身炎症反应和肝外器官衰竭是A C L F 进展的关键因素,因此可用来评估疾病的预后㊂A C L F 是一种动态的综合征,超过50%的患者最终因失代偿事件而死亡[1]㊂短期死亡率是由从A C L F 发病初期到终末期这个临床进程决定的,通常是在发病7天之内[2]㊂随着研究的不断深入,A C L F 评分模型也在持续演变,从终末期肝病模型(M E L D )㊁M E L D -N a ㊁序贯器官衰竭评分(S O F A )直到欧洲慢性肝功能衰竭研究基金会(E u r o p e a n F o u n d a t i o n f o rt h e S t u d y o f C h r o n i cL i v e rF a i l u r e ,E FC l i f )A C L F 评分(C L I F -CA C L F ),评价效能也在不断提升,准确性最高可达75%㊂寻找一个可靠的A C L F 生物标志物是为了能够早期识别并积极治疗预后不良的患者,避免对病死率达到75%~100%的患者采用昂贵的治疗,仅给予临终支持治疗[3],并可能优化现有评分模型㊂各种新型生物标志物是从A C L F 的发病机制着手探索出来的一系列可能具有潜在预后价值的生物活性物质,本文旨在探讨国内外近年来涌现出的各种新型生物学标志物在预测A C L F 中的价值㊂以下将其划分为蛋白质类㊁细胞类㊁核酸类㊁代谢组学四类分别进行阐述㊂1 蛋白质类标志物蛋白质组学是继基因组学研究之后生命科学的前沿学科,对蛋白质组学的研究可以深入地发现和理解㊃339㊃‘临床荟萃“ 2018年11月5日第33卷第11期 C l i n i c a l F o c u s ,N o v e m b e r 5,2018,V o l 33,N o .11Copyright ©博看网. All Rights Reserved.重要的生理㊁病理现象的本质㊂目前肝病蛋白质组学研究旨在寻找用于早期诊断和预后评估的生物标志物和药物作用靶点,探索疾病发生发展的机制㊂1.1炎症反应相关标志物 A C L F的发病机制之一即过度全身炎症反应,也是疾病进展的关键因素,能够体现机体全身炎症反应的程度及发展阶段的生物学标志物无疑对A C L F的早期诊断及预后评估具有重要价值㊂细胞因子及趋化因子是参与炎症反应的重要组成部分㊂D i r c h w o l f等[4]研究发现,促炎因子白细胞介素(I L)-2㊁I L-6㊁I L-8和干扰素(I F N)-γ与A C L F 严重程度显著相关,可能成为A C L F潜在的预后标志物㊂一项旨在探寻可早期评估乙型肝炎病毒相关A C L F(H B V-A C L F)预后的关键炎症因子的研究发现,由慢性乙型病毒性肝炎进展至H B V-A C L F的过程中,I L-10是唯一一个显著降低的细胞因子(P= 0.008),且I L-10的浓度与丙氨酸氨基转移酶(A L T)水平呈显著正相关(r=0.711,P<0.001)㊂因此在慢性乙型病毒性肝炎急性加重期时检测血清I L-10的水平,有可能预测和早期诊断A C L F[5]㊂I L-23通过作用于记忆性T细胞和炎症性巨噬细胞的I L-23受体发挥功能,研究发现H B V-A C L F患者治疗前高水平的I L-23与患者高死亡率相关,可能成为一项提示不良预后的标志物[6]㊂其他一些促炎细胞因子如I L-1β㊁I L-17㊁巨噬细胞释放的凝血活性因子㊁人纤维介素等均可加重肝损伤,促进肝衰竭的进展[7-10]㊂趋化因子可趋化白细胞的迁移,一项有关单核细胞趋化蛋白-1 (M C P-1)的研究发现,该趋化因子在肝衰竭患者中显著升高,与慢性肝病肝组织炎症相关,可作为一项评价肝脏炎症水平的可靠标志物[11]㊂其他如巨噬细胞炎性蛋白3α(M I P-3α)㊁趋化因子10(C X C L10)与慢性乙型病毒性肝炎患者肝脏炎症程度相关[12]㊂胱抑素-C是评估肾小球滤过率的重要指标,其敏感性好,特异性高,也是评估A C L F患者发生急性肾衰竭的重要指标㊂研究证明,血清胱抑素C基线水平可以作为预测A C L F死亡率的独立预后标志物[13]㊂另一项研究发现,血清胱抑素C和总胆红素(T B i L)是与生存率显著相关的两个独立因素,血清胱抑素C联合T B i L所得预后指数P I可以作为早期预测H B V-A C L F短期死亡率的一项新兴指标,其准确性优于C h i l d-T u r c o t t e-P u g h(C T P)㊁M E L D和M E L D-N a评分[14]㊂A r i z a等[15]研究发现,A C L F尿液里中性粒细胞明胶酶相关脂质运载蛋白(N G A L)增加与生存率呈正相关,可以显著提高M E L D评估预后的准确性,该指标是否可以增加C L I F-C A C L F 评分的动态预测效能还有待于进一步研究证实㊂α1酸性糖蛋白是一种急性时相反应蛋白,H B V-A C L F患者α1酸性糖蛋白表达水平显著降低,可能作为预测A C L F发展的一个标志物[16]㊂促甲状腺激素是预测A C L F死亡的独立危险因素,其水平< 0.38I U/m l时,患者累积生存率显著升高[17]㊂终末期肝病表现出各种白蛋白功能紊乱,可能与半胱氨酸-34蛋白的氧化修饰相关,包括不可逆氧化修饰人类n o n m e r c a p t a l b u m i n-2(H N A2),研究证明H N A2可用于预测终末期肝病30天及90天死亡率,R O C 曲线下面积(A U C)显示预测的准确性及特异性均达到80%以上,这标志着H N A2可以成为一种新的慢性肝衰竭的生物学标志物[18]㊂骨桥蛋白(o s t e o p o n t i n,O P N)可能在肝脏祖细胞扩增中起关键作用,并可促进肝癌和急性肝衰竭时肝脏再生[19-22]㊂一项有关O P N预测A C L F患者90天生存率的研究发现,A C L F患者血清O P N显著升高,是与H B V-A C L F预后相关的独立危险因素,可能成为潜在的预后标志物[23]㊂一项旨在探索能够有效预测A C L F预后的潜在血清蛋白生物学标志物的研究发现了6个潜在指标,其中甘露醇受体(m a n n o s e r e c e p t o r,MMR)与免疫应答有关,O P N㊁血红素结合蛋白(h e m o p e x i n, H P X)与炎症反应有关,抗凝血酶Ⅲ(a n t i t h r o m b i n-Ⅲ,A TⅢ)与凝血有关,载脂蛋白CⅡ(a p o l i p o p r o t e i nCⅡ,A P O-CⅡ)㊁高尔基体糖蛋白(g o l g im e m b r a n e p r o t e i n1,G P73)与脂质代谢有关,R O C曲线分析证明上述生物学标志物对H B V-A C L F预后评价有极高的价值[24]㊂应用E L I S A方法检测血清中晚期氧化蛋白产物(A d v a n c e d O x i d a t i o n P r o t e i n P r o d u c t s,A O P P)㊁S100A12㊁HMG B1和可溶性晚期糖基化终末产物受体(s R A G E),结果发现,A C L F组上述指标显著升高,多变量C O X回归分析证明上述指标是不良预后的独立危险因素,可作为重要的预后标志物[25]㊂此外,还有铁蛋白㊁前白蛋白㊁甲胎蛋白㊁A p o A-I等与肝损伤的严重程度相关,可能成为潜在的预后标志物[26-29]㊂肿瘤坏死因子(T N F)-α转化酶(T A C E)也可作为A C L F预后评估的潜在标志物[30]㊂1.2细胞凋亡或坏死相关标志物 A C L F患者肝细胞大量坏死和凋亡,导致肝细胞数量锐减,引起肝衰竭㊂损伤相关的分子模式(D AM P s)即损伤细胞发生凋亡或坏死时所释放大量活性物质或危险信号分子,主要包括高迁移率族蛋白1(HMG B1)㊁热休克蛋白等,HMG B1表达于几乎所有真核细胞,凋亡及坏㊃439㊃‘临床荟萃“2018年11月5日第33卷第11期 C l i n i c a l F o c u s,N o v e m b e r5,2018,V o l33,N o.11Copyright©博看网. All Rights Reserved.死细胞均可以释放,它代表总细胞的死亡㊂血清M30-抗原水平代表细胞凋亡的肝细胞死亡,M-65代表肝细胞总死亡㊂为了评估M30-抗原㊁M65-抗原和HMG B1的预后价值,C a o等[31]研究发现,H B V-A C L F上述三项指标均显著上升,其中M30㊁M65-抗原是3个月短期死亡率的独立预测因子,但HMG B1被认为无预测价值,值得注意的是研究中所建立的M E L D-c e l l d e a t h(M E L D-C D)模型评价效能要高于M E L D㊁M E L D-N a㊁C T P s等现有评分模型㊂1.3免疫反应相关标志物 A C L F患者的典型特征包括易于感染㊁免疫麻痹㊁单核细胞功能缺陷㊂原癌基因酪氨酸激酶(M E R T K)表达于单核细胞和巨噬细胞并促进固有免疫的下调㊂一项研究发现, A C L F组患者外周血㊁肝脏㊁淋巴结中M E R T K表达显著上调,并抑制了针对微生物的固有免疫反应,能够表达M E R T K的免疫调节相关单核㊁巨噬细胞的数量与A C L F的严重度和炎症反应相关[32]㊂T N F-α诱生蛋白8样分子2(T I P E2)是一种维持免疫稳态的负性调控因子,A C L F患者体内T I P E2m R N A高表达,可能通过下调细胞免疫参与A C L F的发病,有潜在的预测A C L F预后的作用[33]㊂免疫分型研究有利于绘制A C L F免疫指纹图谱,有助于我们更好的界定和早期识别那些具有发生脓毒症和高病死率风险的患者,并给予新兴治疗(如M E R T K抑制剂)[3]㊂T o l l样受体(T o l l-l i k er e c e p t o r,T L R)是一类天然免疫受体,可识别结合病原体及其产物,导致炎性介质释放,激活获得性免疫㊂T L R-4是介导内毒素应答的最主要受体,研究发现位于外周血单核细胞中T细胞上的T L R4与H B V-A C L F加重有关,可作为评估预后的标志物[34]㊂胸腺素β4与机体免疫功能㊁创伤愈合等功能密切相关,研究发现胸腺素β4与A C L F患者疾病严重程度相关,可作为评价A C L F患者预后的重要潜在标志物[35]㊂2细胞类标志物免疫功能紊乱是A C L F发病的重要机制之一,机体免疫状态的评估对疾病诊断及预后评价都有积极的意义㊂中性粒-淋巴细胞比值(N e u t r o p h i lt o L y m p h o c y t eR a t i o,N L R)是一项可以反映治疗前患者炎症状况且临床易于获得的生物学指标,L i u 等[36]研究发现,N L R与H B V-A C L F预后相关, N L R越高则预后越差,N L Rɤ2.36预示较低死亡率(灵敏度91.6%,阴性预测值86%),N L R>6.12预示高死亡风险(特异度90.1%,阳性预测值80.3%),因此N L R可作为一项评估A C L F预后的有力指标㊂M o r e a u等[37]也得出了相同结论,认为N L R可以预测A C L F患者90天病死率,若联合S O F A评分㊁是否需要机械通气等指标可以成为颇为有用的预测工具㊂L i n等[38]证实了N L R也可以用于预测肝移植后发生A C L F的不良预后㊂一项有关C D3+T细胞与单核细胞比值(T/M)对A C L F患者预后评估效能的研究显示,随着A C L F进展,患者的T/M进行性下降,T/M可作为一项潜在的预后标志物[39]㊂Z h a n g等[40]研究发现,辅助性T细胞17(T h e l p c e l l17,T h17)在H B V-A C L F患者中显著升高,与疾病的严重程度呈正相关,R O C曲线分析显示,与M E L D-N a㊁C L I F-C A C L F评分相比,T h17细胞频率对预测A C L F患者90天生存率拥有同样的准确性,更重要的是当T h17细胞与上述评分系统联合应用时,其评价效能更高, T h17细胞频率大于5.9%可以作为A C L F预后不良的标志物㊂3核酸类标志物随着基因技术的发展,基因谱逐渐被引入肝脏疾病的研究,尝试从基因角度解释肝脏疾病的发病机制㊁早期诊断及评估预后并提供可能的治疗方法㊂微小R N A(m i-c r o R N A,m i R N A)的高分子稳定性和分布特征以及新兴的快速评估技术使m i R N A成为潜在的肝衰竭标志物㊂D i n g等[41]通过研究H B V-A C L F患者外周血单核细胞微小核糖核酸(m i R N A)的特征,发现了6个极有可能用于预测H B V-A C L F 进展的新兴标志物,包括h s a-m i R-21-5p㊁h s a-m i R-34c-5p㊁h s a-m i R-143-3p㊁h s a-m i R-143-5p㊁h s a-m i R-374a-3p a n dh s a-m i R-542-3p㊂P u等[42]经研究证实m i R N A-146a,m i R N A-150,m i R N A-29a与H B V-A C L F患者生存率正相关,可用于评估此类患者预后㊂Z h e n g等[43]研究发现m i R N A-130a与患者良好预后正相关,它可能成为H B V-A C L F一项重要的潜在预后标志物㊂此外,还有m i R N A-1187㊁m i R N A-150㊁m i R N A-221㊁m i R N A122也可能作为潜在预后评价标志物提高现有评分系统的效能[44-45]㊂由于m i R N A s的定量检测和标准尚未统一,仍需大量研究证实㊂4代谢产物标志物与基因组学和蛋白质组学相比,代谢组学与生理学的联系更加紧密,代谢物反映了细胞所处环境㊂通过对某些代谢产物进行分析,寻找A C L F的生物标记物,在A C L F早期诊断和评价预后方面起重要作用㊂目前用于检测代谢产物的主要技术手段有核磁共振㊁液相色谱-质谱联用技术(L C-M S)㊁色谱分析如高效液相色谱法和气相色谱法(H P L C,G C)等㊂㊃539㊃‘临床荟萃“2018年11月5日第33卷第11期 C l i n i c a l F o c u s,N o v e m b e r5,2018,V o l33,N o.11Copyright©博看网. All Rights Reserved.增强的氧化应激反应是A C L F重要发病机制之一,氧化应激产物可以激活炎症细胞,例如中性粒细胞㊁单核细胞,引发细胞因子级联反应,从而促进器官损伤㊂L i u等[46]利用分光光度法检测发现,A O P P是A C L F患者预后的独立危险因素,A O P Pȡ74.21μm o l/L提示预后不良㊂另外该研究还发现单纯血浆置换可有效清除A O P P,改善患者预后㊂一项采用高效液相色谱-串联质谱法(U P L C/M S)对H B V-A C L F的患者进行代谢物分析研究发现,以下17种代谢产物可能成为潜在的预后标志物,包括溶血性磷脂酰胆碱(L y s o P C)(18:0)㊁L y s o P C(17:0)㊁L y s o P C(16:0)㊁L y s o P C(15:0)㊁L y s o P C(14:0)㊁L-苏氨酸㊁乙酰乙酸㊁磷酸二羟丙酮(18:0)㊁苯丙氨酰基㊁苯丙氨酸㊁结合胆红素㊁P A(20:4(5Z,8Z,11Z, 14Z)e/2:0)㊁m/z=210.05㊁m/z=475.172㊁m/z= 797.3㊁m/z=599.29㊁m/z=258.818㊁m/z= 775.318㊂上述指标仍需大样本研究来验证其临床应用价值[47]㊂此外,二甲基精氨酸㊁甘油酸顺式乌头酸㊁柠檬酸等可能成为潜在的A C L F预后标志物[48]㊂5结论A C L F是一种进展迅速㊁预后极为凶险的疾病,短期死亡率高,只有早期诊断并给予有效治疗才是患者生存的关键㊂我们回顾了近年来涌现出的大量有关A C L F预后的生物学标志物,其中相当一部分表现出了极佳的预测效能,很可能在将来应用于预后评分系统当中,推进现有评分体系的发展,增加预测的准确性㊂然而,上述临床研究样本量普遍较少,未来需要更多设计精良㊁大样本㊁前瞻性临床研究来证明这些预后标志物的评价效能,另外临床检测的易行性以及经济学效益也需要考虑在内㊂除了研究单项标志物,我们更需要关注的是复合标志物与临床评分系统的联合应用,这样可以更好的评价早期治疗效果并具有更出色预后评估效能㊂参考文献:[1] M o r e a uR,J a l a n R,G i n e sP,e ta l.A c u t e-o n-c h r o n i cl i v e rf a i l u r e i sad i s t i n c ts y n d r o m et h a td e v e l o p si n p a t i e n t s w i t ha c u t e d e c o m p e n s a t i o n o f c i r r h o s i s[J].G a s t r o e n t e r o l o g y,2013,144(7):1426-37,1437.[2] G u s t o tT,F e r n a n d e zJ,G a r c i aE,e ta l.C l i n i c a lC o u r s eo fa c u t e-o n-c h r o n i c l i v e r f a i l u r e s y n d r o m e a n d e f f e c t s o np r o g n o s i s[J].H e p a t o l o g y,2015,62(1):243-52. [3] M o o k e r j e eR P.P r o g n o s i sa n db i o m a r k e r s i na c u t e-o n-c h r o n i cl i v e r f a i l u r e[J].S e m i nL i v e rD i s,2016,36(2):127-32.[4] D i r c h w o l f M,P o d h o r z e r A,M a r i n o c M,e t a l.I mm u n ed y s f u n c t i o n i n c i r r h o s i s:D i s t i n c t c y t o k i ne s p h e n o t y p e sa c c o r d i n g t o c i r r h o s i s s e v e r i t y[J].C y t o k i n e,2016,77:14-25.[5] W a n g K,W uZ B,Y e Y N,e ta l.P l a s m ai n t e r l e u k i n-10:al i k e l yp r e d i c t i v em a r k e r f o rh e p a t i t i sBv i r u s-r e l a t e da c u t e-o n-c h r o n i c l i v e r f a i l u r e[J].H e p a tM o n,2014,14(7):e19370.[6] B a oS,Z h e n g J,L i N,e t a l.R o l e o fi n t e r l e u k i n-23i nm o n o c y t e-d e r i v e d d e n d r i t i c c e l l s o f H B V-r e l a t e d a c u t e-o n-c h r o n i c l i v e r f a i l u r e a nd i t s c o r re l a t i o nw i t h t h e s e v e r i t y of l i v e rd a m a g e[J].C l i n Re s H e p a t o lG a s t r o e n t e r o l,2017,41(2):147-155.[7] C h e nE Q,B a i L,G o n g D Y,e t a l.E m p l o y m e n t o f d i g i t a l g e n ee x p r e s s i o n p r of i l i ng t o i d e n t i f y p o t e n t i a l p a th o g e ni c a n dt h e r a p e u t i c t a r g e t so f f u l m i n a n th e p a t i cf a i l u r e[J].JT r a n s lM e d,2015,13:22.[8] Z h a n g J Y,Z h a n g Z,L i n F,e ta l.I n t e r l e u k i n-17-p r o d u c i n gC D4(+)T c e l l si n c r e a s e w i t hs e v e r i t y o fl i v e rd a m a g ei np a t i e n t sw i t hc h r o n i ch e p a t i t i sB[J].H e p a t o l o g y,2010,51(1):81-91.[9] W a s m u t h H E,T a c k eF,T r a u t w e i n C.C h e m o k i n e s i nl i v e ri n f l a mm a t i o n a n d f i b r o s i s[J].S e m i nL i v e rD i s,2010,30(3):215-225.[10] K i m H Y,J h u n J Y,C h oM L,e t a l.I n t e r l e u k i n-6u p r e g u l a t e sT h17r e s p o n s e v i a m T O R/S T A T3p a t h w a y i n a c u t e-o n-c h r o n i c h e p a t i t i sB l i v e r f a i l u r e[J].JG a s t r o e n t e r o l,2014,49(8):1264-1273.[11] M a r s i l l a c hJ,B e r t r a nN,C a m p s,e t a l.T h e r o l e o f c i r c u l a t i n gm o n o c y t ec h e m o a t t r a c t a n t p r o t e i n-1a sa m a r k e ro fh e p a t i ci n f l a mm a t i o n i n p a t i e n t s w i t hc h r o n i cl i v e rd i s e a s e[J].C l i nB i o c h e m,2005,38(12):1138-1140.[12] R o t hG A,F a y b i kP,H e t zH,e t a l.M C P-1a n d M I P3-a l p h as e r u ml e v e l si n a c u t el i v e rf a i l u r ea n d m o l e c u l a ra d s o r b e n t r e c i r c u l a t i n g s y s t e m(MA R S)t r e a t m e n t:a p i l o ts t u d y[J].S c a n d JG a s t r o e n t e r o l,2009,44(6):745-751. [13] M a r k w a r d tD,H o l d t L,S t e i bC,e t a l.P l a s m a c y s t a t i nC i s ap r e d i c t o r o fr e n a ld y s f u n c t i o n,a c u t e-o n-c h r o n i c l i v e r f a i l u r e,a n d m o r t a l i t y i n p a t i e n t s w i t h a c u t e l y d e c o m p e n s a t e dl i v e rc i r r h o s i s[J].H e p a t o l o g y,2017,66(4):1232-1241.[14] W a nZ,W uY,Y i J,e t a l.C o m b i n i n g s e r u mc y s t a t i nC w i t ht o t a lb i l i r u b i n i m p r o v e s s h o r t-t e r m m o r t a l i t y p r e d i c t i o n i np a t i e n t sw i t h H B V-r e l a t e da c u t e-o n-c h r o n i cl i v e rf a i l u r e[J].P L o SO n e,2015,10(1):e0116968.[15] A r i z aX,G r a u p e r aI,C o l l M,e ta l.N e u t r o p h i l g e l a t i n a s e-a s s o c i a t e dl i p o c a l i ni sab i o m a r k e ro fac u t e-o n-c h r o n i cl i v e rf a i l u r e a n d p r og n o s i s i n c i r rh o si s[J].JH e p a t o l,2016,65(1):57-65.[16] R e n F,C h e n Y,W a n g Y,e t a l.C o m p a r a t i v e s e r u mp r o t e o m i c a n a l y s i s o f p a t i e n t s w i t h a c u t e-o n-c h r o n i c l i v e rf a i l u r e:a l p h a-1-a c i dg l y c o p r o t e i n m a y b eac a n d i d a t e m a r k e rf o r p r og n o s i s o fh e p a ti t i sBv i r u s i n f e c t i o n[J].JV i r a lH e p a t,2010,17(11):816-824.[17] W uY,Y o uS,Z a n g H,e ta l.U s e f u l n e s so f s e r u mt h y r o i d-s t i m u l a t i o n h o r m o n e(T S H)a s a p r o g n o s t i ci n d i c a t o rf o ra c u t e-o n-c h r o n i c l i v e r f a i l u r e[J].A n nH e p a t o l,2015,14(2):218-224.[18]S t a u b e r R E,S p i n d e l b o e c k W,H a a s J,e t a l.H u m a nn o n m e r c a p t a l b u m i n-2:an o v e l p r o g n o s t i c m a r k e ri nc h r o n i c l i v e r f a i l u r e[J].T h e rA p h e rD i a l,2014,18(1):74-78. [19] L i uY,C a oL,C h e nR,e t a l.O s t e o p o n t i n p r o m o t e sh e p a t i cp r o g e n i t o r c e l l e x p a n s i o n a n d t u m o r i g e n i c i t y v i a a c t i v a t i o n o fβ-c a t e n i n i nm i c e[J].S t e m C e l l s,2015,33(12):3569-3580.[20] L e eS H,P a r k J W,W o o S H,e t a l.S u p p r e s s i o n o f o s t e o p o n t i ni n h i b i t s c h e m i c a l l y i n d u c e dh e p a t i c c a r c i n o g e n e s i sb y i n d u c t i o n㊃639㊃‘临床荟萃“2018年11月5日第33卷第11期 C l i n i c a l F o c u s,N o v e m b e r5,2018,V o l33,N o.11Copyright©博看网. All Rights Reserved.o f a p o p t o s i s i n m i c e[J].O n c o t a r g e t,2016,7(52):87219-87231.[21]S r u n g a r a m P,R u l eJ A,Y u a n H J,e ta l.A c u t e l i v e r f a i l u r es t u d yg r o u p:p l a s m ao s t e o p o n t i ni n a c u t el i v e rf a i l u r e[J].C y t o k i n e,2015,73(2):270-276.[22] T a j i r iT,T a t eG,K u n i m u r aT,e t a l.O s t e o p o n t i n e x p r e s s i o ni n p r o l i f e r a t e d b i l e d u c t u l e s:T h e c o r r e l a t i o nw i t h l i v e r d a m a g ei n f u l m i n a n t h e p a t i t i s[J].D i g D i sS c i,2005,50(1):188-195.[23] L i uL,L u J,Y eC,e t a l.S e r u mo s t e o p o n t i n i s a p r e d i c t o r o fp r o g n o s i sf o r H B V-a s s o c i a t e d a c u t e-o n-c h r o n i cl i v e rf a i l u r e[J].B i o m e dR e p,2018,8(2):166-171.[24] Z h o uN,W a n g K,F a n g S,e ta l.D i s c o v e r y o fa p o t e n t i a lp l a s m a p r o t e i n b i o m a r k e r p a n e lf o r a c u t e-o n-c h r o n i c l i v e rf a i l u r e i n d u c e db y h e p a t i t i sBv i r u s[J].F r o n tP h y s i o l,2017,8:1009.[25] C a i J,H a nT,N i eC,e t a l.B i o m a r k e r so fo x i d a t i o ns t r e s s,i n f l a mm a t i o n,n e c r o s i s a n d a p o p t o s i s a r e a s s o c i a t e d w i t hh e p a t i t i sB-r e l a t e da c u t e-o n-c h r o n i c l i v e r f a i l u r e[J].C l i nR e sH e p a t o lG a s t r o e n t e r o l,2016,40(1):41-50.[26] H u a n g L,L i J,Y a nJ J,e ta l.P r e a l b u m i ni s p r e d i c t i v ef o rp o s t o p e r a t i v el i v e ri n s u f f i c i e n c y i n p a t i e n t su n d e r g o i n g l i v e r r e s e c t i o n[J].W o r l dJ G a s t r o e n t e r o l,2012,18(47):7021-7025.[27] K a k i s a k a K,K a t a o k a K,O n o d e r a M,e t a l.A l p h a-f e t o p r o t e i n:A b i o m a r k e rf o rt h er e c r u i t m e n to f p r og e n i t o rc e l l s i nt h e l i v e r i n p a t i e n t sw i t ha c u t e l i v e r i n j u r y o nf a i l u r e[J].H e p a t o lR e s,2015,45(10):E12-E20.[28] T s a i MH,P e n g Y S,C h e n Y C,e t a l.L o w s e r u mc o n c e n t r a t i o n o fa p o l i p o p r o t e i n A-Ii sa ni nd i c a t o ro f p o o rp r o g n o s i si n c i r r h o t i c p a t i e n t s w i t h s e v e r e s e p s i s[J].JH e p a t o l,2009,50(5):906-915.[29] W u J,C h e nL,C h e n Y,e ta l.S e r u mf e r r i t i nc o n c e n t r a t i o np r e d i c t s m o r t a l i t y i n p a t i e n t s w i t h h e p a t i t i s B v i r u s-r e l a t e da c u t e o nc h r o n i c l i v e rf a i l u r e[J].A r c h M e d R e s,2014,45(3):251-256.[30] W a n g Z L,G a o S,L iL,D e m e t h y l a t i o n o ft u m o rn e c r o s i sf a c t o r-αc o n v e r t i ng e n z y m e p r e d i c t s p o o r p r o g n o s i s i n a c u t e-o n-c h r o n i c h e p a t i t i s B l i v e r f a i l u r e[J].C l i n R e s H e p a t o lG a s t r o e n t e r o l,2016,40(4):457-464.[31] C a oZ,L i F,X i a n g X,e t a l.C i r c u l a t i n g c e l l d e a t h b i o m a r k e r:g o o d c a n d i d a t e s o f p r o g n o s t i c i n d i c a t o r f o r p a t i e n t s w i t hh e p a t i t i sBv i r u s r e l a t e da c u t e-o n-c h r o n i c l i v e r f a i l u r e[J].S c iR e p,2015,5:14240.[32] B e r n s m e i e rC,P o p O T,S i n g a n a y a g a m A,e t a l.P a t i e n t sw i t ha c u t e-o n-c h r o n i c l i v e r f a i l u r e h a v e i n c r e a s e d n u mb e r s o fr e g u l a t o r y i mm u n e c e l l s e x p r e s s i n g t h e r e c e p t o r t y r o s i n ek i n a s eM E R T K[J].G a s t r o e n t e r o l o g y,2015,148(3):603-615.[33] W a n g L Y,F a n Y C,Z h a oJ,e ta l.E l e v a t e de x p r e s s i o no ft u m o u r n e c r o s i s f a c t o r-α-i n d u c e d p r o t e i n8(T N F A I P8)-l i k e2m R N Ai n p e r i p h e r a l b l o o dm o n o n u c l e a r c e l l s i s a s s o c i a t e dw i t hd i se a s e p r o g r e s s i o nof a c u t e-o n-c h r o n i c h e p a t i t i sB l i v e r f a i l u r e[J].JV i r a lH e p a t,2014,21(1):64-73.[34] X uC L,H a o Y H,L u Y P,e ta l.U p r e g u l a t i o n o ft o l l-l i k er e c e p t o r4o n T c e l l si n P B M C si sa s s o c i a t e d w i t h d i s e a s ea g g r a v a t i o no fH B V-r e l a t e da c u t e-o n-c h r o n i c l i v e r f a i l u r e[J].JH u a z h o n g U n i vS c iT e c h n o l o g M e dS c i,2015,35(6):910-915.[35] H a nT,L i uY,L i uH,e t a l.S e r u mt h y m o s i nb e t a4l e v e l s i np a t i e n t sw i t hh e p a t i t i s Bv i r u s-r e l a t e d l i v e r f a i l u r e[J].W o r l d JG a s t r o e n t e r o l,2010,16(5):625-630.[36] L i u H,Z h a n g H,W a nG,e t a l.N e u t r o p h i l-l y m p h o c y t e r a t i o:an o v e l p r e d i c t o r f o r s h o r t-t e r m p r o g n o s i s i na c u t e-o n-c h r o n i ch e p a t i t i sB l i v e r f a i l u r e[J].JV i r a lH e p a t,2014,21(7):499-507.[37] M o r e a u N,W i t t e b o l e X,F l e u r y Y,e ta l.N e u t r o p h i l-t o-l y m p h o c y t e r a t i o p r e d i c t s d e a t h i n a c u t e-o n-c h r o n i c l i v e r f a i l u r ep a t i e n t sa d m i t t e dt ot h ei n t e n s i v ec a r eu n i t:ar e t r o s p e c t i v ec o h o r t s t ud y[J].S h o c k,2018,49(4):385-392.[38] L i nB Y,Z h o uL,G e n g L,e t a l.H i g hn e u t r o p h i l-l y m p h o c y t er a t i o i n d i c a t e s p o o r p r o g n o s i s f o r a c u t e-o n-c h r o n i c l i v e r f a i l u r ea f t e r l i v e r t r a n s p l a n t a t i o n[J].W o r l dJG a s t r o e n t e r o l,2015,21(11):3317-3324.[39]S h i F,Z h a n g J Y,Z e n g Z,e t a l.S k e w e dr a t i o sb e t w e e nC D3(+)Tc e l l s a n dm o n o c y t e s a r e a s s o c i a t e dw i t h p o o r p r o g n o s i si n p a t i e n t sw i t hH B V-r e l a t e d a c u t e-o n-c h r o n i c l i v e r f a i l u r e[J].B i o c h e m B i o p h y sR e sC o mm u n,2010,402(1):30-36.[40] Z h a n g G L,Z h a n g T,Z h a oQ Y,e t a l.T h17c e l l so v e r5.9%a ta d m i s s i o ni n d i c a t e p o o r p r o g n o s i si n p a t i e n t s w i t h H B V-r e l a t e d a c u t e-o n-c h r o n i c l i v e r f a i l u r e[J].M e d i c i n e(B a l t i m o r e),2018,97(40):e12656.[41] D i n g W,X i n J,J i a n g L,e t a l.C h a r a c t e r i s a t i o no f p e r i p h e r a lb l o o dm o n o n uc l e a r c e l lm i c r o R N Ai nh e p a t i t i sB-r e l a t ed a c u t e-o n-c h r o n i c l i v e r f a i l u r e[J].S c i R e p,2015,5:13098.[42] P uC,J i a n g C,L a n g L,e t a l.C o m b i n a t i o n o fM i c r o R N A s a n dC y t o k i n e s:a M e t h o d f o r B e t t e r E v a l u a t i o n o f A c u t e-o n-C h r o n i cL i v e rF a i l u r e[J].C l i nL a b,2018,64(3):247-256.[43] Z h e n g Q F,Z h a n g J Y,W u J S,e t a l.U p r e g u l a t i o n o fm i R N A-130a R e p r e s e n t s G o o d P r o g n o s i s i n P a t i e n t s W i t h H B V-R e l a t e dA c u t e-o n-C h r o n i cL i v e rF a i l u r e:A P r o s p e c t i v eS t u d y[J].M e d i c i n e(B a l t i m o r e),2016,95(6):e2639. [44] Y uD S,A n F M,G o n g B D,e ta l.T h er e g u l a t o r y r o l eo fm i c r o R N A-1187i n T N F-α-m e d i a t e d h e p a t o c y t ea p o p t o s i si na c u t e l i v e r f a i l u r e[J].I n t JM o lM e d,2012,29(4):663-668.[45] Y u a n Q,L o y a K,R a n iB.M i c r o R N A-221o v e r e x p r e s s i o na c c e l e r a t e sh e p a t o c y t e p r o l i f e r a t i o n d u r i n g l i v e rr e g e n e r a t i o n[J].H e p a t o l o g y,2013,57(1):299-310.[46] L i uH,H a nT,T i a nJ.M o n i t o r i n g o x i d a t i v es t r e s s i na c u t e-o n-c h r o n i c l i v e r f a i l u r eb y a d v a n c e do x i d a t i o n p r o t e i n p r o d u c t s[J].JH e p a t o lR e s,2012,42(2):171-180.[47] M o o k e r j e eR P,D a v i e sN A,D a l t o nR N,e t a l.E v a l u a t i o no ft h e b i o m a r k e r,d i m e t h y l a r g i n i n e s c o r e-i s c h e m i a m o d i f i e da lb u m i n r a t i o(D A S I MA R)t o i d e n t i f y r i s ko f p r o g r e s s i o nt oa c u t e o n c h r o n i c l i v e r f a i l u r e a n dm o r t a l i t y(A C L F)i n p a t i e n t sw i t hd e c o m p e n s a t e d c i r r h o s i s[J].H e p a t o l o g y,2007,46(4): 615A.[48] M a oY,H u a n g X,Y u K,e ta l.M e t a b o n o m i ca n a l y s i so fh e p a t i t i s B v i r u s-i n d u c e d l i v e r f a i l u r e:i d e n t i f i c a t i o n o fp o t e n t i a l d i a g n o s t i c b i o m a r k e r s b y f u z z y s u p p o r t v e c t o rm a c h i n e[J].JZ h e j i a n g U n i vS c i B,2008,9(6):474-481.收稿日期:2018-11-27编辑:张卫国㊃739㊃‘临床荟萃“2018年11月5日第33卷第11期 C l i n i c a l F o c u s,N o v e m b e r5,2018,V o l33,N o.11Copyright©博看网. All Rights Reserved.。
Nephrol. Dial. Transplant.
Nephrol Dial Transplant (2009)1321 13doi:10.1093/ndt/gfp500Recurrence of nephrotic syndrome after transplantation in a mixed population of children and adults:course of glomerular lesions and value of the Columbia classification of histological variants of focal and segmental glomerulosclerosis (FSGS)Guillaume Canaud 1,2,Daniel Dion 3,Julien Zuber 1,2,Marie-Claire Gubler 4,Rebecca Sberro 1,2,Eric Thervet 1,2,Renaud Snanoudj 1,Marina Charbit 5,R ´e mi Salomon 2,5,Frank Martinez 1,Christophe Legendre 1,2,Laure-Helene Noel 3and Patrick Niaudet 2,51Department of Kidney Transplantation,Necker Hospital,149rue de S`e vres,75015,Paris,2Universit´e Paris Descartes,Rue del’Ecole de M´e decine,3Department of Pathology,4Institut National de la Sant´e et de la Recherche M´e dicale U574and 5Department of Pediatrics Nephrology,Necker Hospital,149rue de S`e vres,75015,Paris,France Correspondence and offprint requests to :Guillaume Canaud;E-mail:guillaume.canaud@nck.aphp.frAbstractIntroduction.Recurrence of nephrotic-range proteinuria in patients with idiopathic nephrotic syndrome (INS)and focal and segmental glomerulosclerosis (FSGS)on native kidneys is associated with poor graft survival.Identifica-tion of risk factors for recurrence is therefore an important issue.In 2004,Columbia University introduced a histo-logical classification of FSGS that identifies five mutually exclusive variants.In non-transplant patients,the Columbia classification appears to predict the outcome and response to treatment better than clinical characteristics alone.How-ever,the predictive value of this classification to assess the risk of recurrence after transplantation has not been addressed.Methods.We retrospectively studied 77patients with INS and FSGS on native kidneys who underwent renal trans-plantation.Of these,42recipients experienced recurrence of nephrotic range proteinuria.Results.At time of recurrence,minimal-change disease (MCD)was the main histological feature.On serial biop-sies,the incidence of MCD decreased over time,while the incidence of FSGS variants increased.The variant type ob-served in the native kidneys was not predictive of either recurrence or type of FSGS seen on the allograft.Patients with complete and sustained remission did not developed FSGS.Conclusion.In conclusion,the Columbia classification is of no help in predicting recurrence after renal transplan-tation or histological lesions in the case of recurrence of proteinuria.Keywords:FSGS;histopathology;kidney transplantation;recurrenceIntroductionSteroid-resistant idiopathic nephrotic syndrome (INS)in native kidneys is frequently associated with progression to end-stage renal disease (ESRD)[1–3].In most patients,ini-tial renal biopsy shows focal and segmental glomeruloscle-rosis (FSGS).However,in early stages,FSGS and minimal-change disease (MCD)are indistinguishable at the clinical level [4–6].Serial renal biopsies indicate that some patients with minimal changes on initial renal biopsy develop FSGS during the course of the disease.This was observed in 60%of cases in a series of 48patients with steroid-resistant min-imal change [7].After kidney transplantation,recurrence of nephrotic-range proteinuria is observed in 30%of cases and is associated with poor graft survival [8–14].Identification of risk factors for recurrence is an important issue.In 2004,Columbia University introduced a histologi-cal classification of FSGS that identifies five mutually exclusive variants:perihilar (PH),cellular (CELL),tip le-sion (TIP),collapsing (COL)and not otherwise specified (NOS)[15].Since then,several studies have correlated re-nal prognosis with histological features in native kidneys.In non-transplant patients,the Columbia variant seems to pre-dict outcome and response to treatment better than clinical characteristics alone.TIP seems to be the most steroid-sensitive variant,with COL being the least favourable vari-ant [16–20].The impact of this classification to predict the FSGS variant in the transplant has been recently studied [21].In one report,Ijpelaar et al.concluded that,in most cases,the FSGS variant observed in the transplant kidney was the same as in the native kidneys,suggesting that the histological variant of the native kidney could predict the variant observed in patients with recurrence.However,the predictive value of this classification to assess the risk ofC The Author 2009.Published by Oxford University Press [on behalf of ERA-EDTA].All rights reserved.For Permissions,please e-mail:journals.permissions@– Advance Access publication 22September 200928 by guest on October 14, 2013/Downloaded fromFig.1.FSGS variants in native kidneys(NOS,n=38;PH,n=6;TIP,n=6;CELL,n=17;COL,n=10).recurrence after transplantation and graft outcome has never been addressed.We investigated in a large series whether the Columbia classification of FSGS variants provides a relevant indicator for the risk of proteinuria recurrence af-ter renal transplantation and the type of variant in the case of recurrence.Material and methodsKidney transplant recipients with INS and FSGS on native kidneys and pro-gression to ESRD were eligible for inclusion.Patients with familial history of proteinuria or secondary forms of FSGS were excluded,i.e.ischaemia-related podocyte injury,sickle cell disease,renal hypoplasia,thrombotic microangiopathy,reflux nephropathy,human immunodeficiency virus and genetic disorders when a test was available.Patients with non-adequate biopsies were also excluded(i.e.the biopsy sample containing fewer than five glomeruli).Our study population included children(n=56)and adults(n=21) (age>16years).There were40males and37females.The77patients included in the study received a sequential quadruple immunosuppression including induction therapy(thymoglobulin,n=47;basiliximab,n=30), corticosteroids,a calcineurin inhibitor(cyclosporine,n=65;tacrolimus, n=12),azathioprine(n=53)or mycophenolate mofetil(n=24).Seventy-one patients had received a first kidney transplant;4patients had received a second transplant and2a third one.Kidney allografts were obtained from a deceased donor in69cases and from a living-related donor in8cases.No patient received pre-emptive treatment for recurrence(i.e.cyclosporine A IV or pre-operative plasmapheresis).Recurrence was defined by massive proteinuria in the nephrotic range (>3g/day for adults or>50mg/kg/day for children),with normal glomeruli and without evidence of acute or chronic rejection,glomerular deposits or allograft glomerulopathy on initial kidney biopsy.Cameron’s classification was applied to define time of recurrence:immediate(<48 h),early(<3months)and late recurrence(>3months)[10].Transplant biopsies were performed either when indicated by protein-uria and/or acute renal failure,or routinely as screening biopsies at3and 12months post-transplant.Native and transplant kidney biopsies were pro-cessed following the international recommendations for light microscopy and immunofluorescence.The median number of glomeruli per biopsy was 13.7±4.6(range6–24).Histology slides were stained with haematoxylin and eosin,periodic acid–Schiff,Masson trichrome and methenamine–silver.For each biopsy,FSGS variant was evaluated according to the Columbia classification[15].The five FSGS variant definitions are sum-marized in Figure1.All native and transplant biopsies were reviewed by three senior pathologists.As electron microscopy is not of current routine practice in France,we were able to describe foot process fusion in only one case.Nevertheless,patients with proteinuria in the nephrotic range and no features of FSGS variants on kidney biopsy were classified as having MCD.ResultsFrom January1984to December2007,77patients with INS and biopsy-proven FSGS on native kidneys received a renal transplant.Patients were classified in two categories according to whether proteinuria recurred in the nephrotic range(R group,n=42)or not(NR group,n=35).The demographic characteristics of the patients are summarized in Tables1and2.The mean age at onset of nephrotic syndrome was10.4±10years and9.2±6.9years for patients in the NR group and in the R group,respectively (P=NS).The delay between the onset of disease and ESRD was4.8±2.3years in the NR group and3.9±3.3years in the R group(P=NS).There was no difference in the immunosuppressive regimen between the two groups.Four patients in each group received a kidney from a living donor. In the NR group,all patients were genotyped for NPHS1and NPHS2,and none of them had mutation.In the R group, 30/42patients were genotyped for NPHS1and NPHS2. T wo patients had a heterozygous variant,polymorphism (pA242V)[22].On native kidney biopsies,MCD was initially observed in10(children exclusively)out of77cases,and repeat re-nal biopsies showed the development of FSGS in all these patients.All variants were present on native kidneys.The1322G.Canaud et al.Table1.Characteristics of patients in the NR groupAge at onset Delay betweenof proteinuria FSGS variant on Number of onset and Patients(years)native kidney glomeruli ESRD(years) 10.8CELL107211COL14 1.633MCD then NOS16245TIP11852NOS235610MCD then CELL1627 3.3NOS12882COL9 2.593NOS2241011CELL135112COL147120.3MCD then NOS8 1.8132NOS256143CELL177150.5NOS117165CELL192171NOS294186TIP254192NOS142204CELL166211NOS119226NOS1092318MCD then PH1342412NOS2062524MCD then NOS2212622PH1882731TIP97.22814CELL9 2.42917CELL743029COL11 3.13119NOS12 3.73216NOS1543334CELL10 5.53428NOS953518NOS9 4.5Mean±SD10.4±1014±5.6 4.8±2.3FSGS variant observed on native kidney biopsies was NOS in38cases(49.3%),CELL in17cases(22.2%),COL in 10cases(12.9%),PH in6cases(7.8%)and TIP in6cases (7.8%).There were no differences in FSGS variant repre-sentation between R and NR groups(Figure2).After kidney transplantation,recurrence of proteinuria occurred immediately in32/42cases(children n=28, adults n=4),early in9/42cases(children n=4,adults n=5)and late in1/42case(adult n=1).The treatment for recurrence consisted of intravenous(IV)cyclosporine A(CsA)+plasmapheresis(PP)in10cases,CsA IV+PP+ a high dose of steroids in3cases,CsA IV alone in10 cases,oral CsA+PP in3cases,oral CsA alone in8cases, oral CsA+PP+rituximab in1case,cyclophosphamide+ PP in1case,CsA IV+PP+rituximab in4cases,CsA IV+enalapril in1case and immunoadsorption in1case (Table2).The distribution of glomerular lesions in transplanted kidneys with recurrence of INS(group R,n=42)is sum-marized in Table2and Figure2.MCD was the main histo-logical feature observed in32out of33biopsies performed early after recurrence,D6to D60days after transplantation. Only one patient had already developed and FSGS lesion (Table2:patient38,PH variant on Day15).At Month3,an Fig.2.Distribution of FSGS variants on a native kidney of patients with or without recurrence of proteinuria in the nephrotic range.FSGS lesion was observed in11out of39cases.At Month 12,FSGS lesion was observed in14out of37cases.Sev-enteen of the42patients went into complete and persistent remission,and none of them developed FSGS(2patients1323Variants of FSGS and risk of recurrenceT a b l e 2.C o u r s e o f F S G S v a r i a n t s o n k i d n e y b i o p s i e s p e r f o r m e d i n t r a n s p l a n t r e c i p i e n t s w i t h r e c u r r e n c e o f p r o t e i n u r i a i n t h e n e p h r o t i c r a n g eT r a n s p l a n t D e l a y b i o p s y a t T r a n s p l a n t T r a n s p l a n t A g e a t b e t w e e n t i m e o f b i o p s y o n b i o p s y o n o n s e t o f F S G S v a r i a n t o n o n s e t a n d r e c u r r e n c e M o n t h 3M o n t h 12p r o t e i n u r i a n a t i v e k i d n e y (n u m b e r E S R D T i m e o f R e s p o n s e t o E v o l u t i o n a f t e r (n u m b e r o f (n u m b e r o f (n u m b e r o f P a t i e n t s (y e a r s )o f g l o m e r u l i )(y e a r s )r e c u r r e n c e T r e a t m e n tt r e a t m e n t t r a n s p l a n t a t i o n g l o m e r u l i )g l o m e r u l i )g l o m e r u l i )O t h e r b i o p s y18N O S (n =12)2D 0C s A I V +P PP a r t i a l T r a n s p l a n t e c t o m y M 12M C D M 1(n =22)M C D (n =12)M C D (n =14)22N O S (n =12)4D 0C s A I V +P PN o r e m i s s i o n T r a n s p l a n t e c t o m y M 1M C D M 1(n =16)X X 36M C D (n =14)t h e n C E L L (n =8)7D 0C s A I VP a r t i a l E S R D 9y e a r sM C D M 1(n =15)M C D (n =9)M C D (n =21)C O L M 24,N O S M 9641.5N O S (n =9)1.5D 0C s A o r a lN o r e m i s s i o n T r a n s p l a n t e c t o m y M 3M C D M 1(n =18)M C D (n =13)X510N O S (n =18)2D 0I m m u n o a d s o r p t i o nC o m p l e t e f o l l o w e d b y r e l a p s e T r a n s p l a n t e c t o m y M 48M C D D 15(n =7)M C D (n =15)M C D (n =14)T r a n s p l a n t e c t o m y M 48C O L62C O L (n =17)3D 0C s A I V +P PC o m p l e t e a n d p e r s i s t e n t M CD D 10(n =12)M C D (n =7)M C D (n =18)M C D M 6270.5C E L L (n =11)2D 3C s A I V C o m p l e t e f o l l o w e d b y r e l a p s e a t M 36T r a n s p l a n t e c t o m y M 60M C D M 1(n =14)C E L L (n =8)C E L L (n =9)C E L L M 36,T r a n s p l a n t e c -t o m y M 60N O S82M C D (n =20)t h e n P H (n =6)7D 0C s A I V +P PC o m p l e t e a n d p e r s i s t e n t M CD (n =12)M C D (n =21)91.3N O S (n =8)5D 0C s A I VC o m p l e t e a n d p e r s i s t e n t M CD (n =24)M C D (n =10)M C D M 108101.5N O S (n =10)8D 0C s A I V +P P +R i t u x i m a b C o m p l e t e a n d p e r s i s t e n t M C D D 14(n =12)M C D (n =12)M C D (n =18)M C D M 36112T I P (n =12)12D 0C s A I VC o m p l e t e a n d p e r s i s t e n t M CD D 15(n =19)M C D (n =15)M C D (n =9)128M C D (n =14)t h e n N O S (n =7)3M 6C s A o r a l P a r t i a lD i e d a t M 144M C D M 6(n =12)M C D (n =9)M C D M 36133N O S (n =8)17D 0C s A I V +P P C o m p l e t e a n d p e r s i s t e n t M C D M 1(n =22)M C D (n =18)M C D (n =8)146C E L L (n =20)4D 0C s A o r a l P a r t i a lT r a n s p l a n t e c t o m y M 36M C D (n =11)M C D (n =14)T r a n s p l a n t e c t o m y M 36N O S 152.1N O S (n =15)3.2D 1C s A I V +e n a l a p r i l C o m p l e t e a n d p e r s i s t e n t M C D (n =7)M C D (n =11)M C D M 96169C O L (n =7)4D 20C s A I V +P P N o r e m i s s i o nT r a n s p l a n t e c t o m y M 60M C D D 10(n =24)M C D (n =11)N O S (n =6)T r a n s p l a n t e c t o m y M 60N O S 178N O S (n =9)7D 0C s A o r a lN o r e m i s s i o nT r a n s p l a n t e c t o m y M 9M C D M 1(n =21)N O S (n =9)X T r a n s p l a n t e c t o m y M 9N O S 187N O S (n =11)4D 1C s A I V +P PC o m p l e t e f o l l o w e d b y r e l a p s e a t M 48M CD (n =18)M C D (n =11)M C D M 48,M C D M 961910M C D (n =11)t h e n N O S (n =12)0.4D 1C s A I VC o m p l e t e f o l l o w e d b y r e l a p s e a t M 9E S RD M 96M C D D 21(n =20)M C D (n =9)C O L (n =14)C O L M 9203N O S (n =14)9D 8C s A I V +P P +r i t u x i m a bC o m p l e t e a n d p e r s i s t e n tM C D M 1(n =12)M C D (n =15)M C D (n =9)1324G.Canaud et al.215N O S (n =16)5D 0C s A I V C o m p l e t e a n d p e r s i s t e n t M C D (n =20)M C D (n =12)M C D 5y e a r s220.5N O S (n =10)2D 3C s A I VC o m p l e t e a n d p e r s i s t e n t M CD D 22(n =11)M C D (n =7)M C D (n =14)235C O L (n =9)8D 1C s A I V C o m p l e t e a n d p e r s i s t e n t M C D D 10(n =9)M C D (n =24)M C D (n =11)245CE L L (n =10)5D 0C s A I V C o m p l e t e f o l l o w e d b y r e l a p s e o n M 24M C D M 1(n =13)CE L L (n =12)C E L L (n =9)C E L L M 242512C O L (n =11)1D 0C s A +P PC o m p l e t e a n d p e r s i s t e n t M CD (n =14)M C D (n =10)265N O S (n =18)2D 0C s A I V +P P N o r e m i s s i o nM C D M 1(n =19)C O L (n =6)C O L (n =9)C O L M 242727C E L L (n =20)5D 0C s A I V +P P C o m p l e t e a n d p e r s i s t e n t M C D D 8(n =11)M C D (n =9)M C D (n =19)2821N O S (n =16)2D 3C s A I V +P P C o m p l e t e a n d p e r s i s t e n t M C D D 6(n =23)M C D (n =14)M C D (n =15)2919C E L L (n =12)5D 0C s A o r a l N o r e m i s s i o nM C D D 10(n =18)M C D (n =16)N O S (n =18)3014C O L (n =11)3D 0C s A I V +P P +h i g h d o s e s t e r o i d s C o m p l e t e a n d p e r s i s t e n t M C D D 8(n =14)M C D (n =20)M C D (n =11)3119C E L L (n =9)1.5D 4C s A o r a l +P P P a r t i a l r e m i s s i o nM C D D 17(n =18)T I P (n =13)P H (n =12)3212P H (n =7)2D 0C s A o r a l N o r e m i s s i o n T r a n s p l a n t e c t o m yM 6M C D D 12(n =16)C O L (n =17)XT r a n s p l a n t e c t o m y M 6C O L3320T I P (n =9)2D 2C s A o r a l P a r t i a l r e m i s s i o n M C D D 10(n =11)M C D (n =16)N O S (n =14)3412C O L (n =11)1.6D 0C s A I V +P P +h i g h d o s e s t e r o i d s C o m p l e t e a n d p e r s i s t e n t M C D (n =20)M C D (n =22)3516C E L L (n =13)0.3D 4C s A I V +P P +r i t u x i m a b N o r e m i s s i o nT r a n s p l a n t e c t o m yM 36M C D D 60(n =10)M C D (n =19)N O S (n =12)T r a n s p l a n t e c t o m y M 36C O L368M C D (n =11)t h e n N O S (n =16)1D 0C s A o r a l +P P +r i t u x i m a b N o r e m i s s i o nP H (n =14)P H (n =16)3717P H (n =9)3D 0C s A o r a lN o r e m i s s i o nC E L L (n =11)C E L L (n =20)3812N O S (n =21)1D 1C s A o r a l +P P P a r t i a lP H D 15(n =17)P H (n =14)P H (n =9)3918T I P (n =15)3D 8C s A I V +P P +h i g h d o s e s t e r o i d s C o m p l e t e a n d p e r s i s t e n t M C D D 10(n =19)M C D (n =17)M C D (n =14)4016P H (n =14)1.5D 1C s A I VN o r e m i s s i o nT r a n s p l a n t e c t o m y M 6M C D D 11(n =21)C O L (n =10)XT r a n s p l a n t e c t o m y M 6C O L4122N O S (n =17)4D 10C s A o r a l +P PN o r e m i s s i o nM C D D 12(n =18)C O L (n =11)C O L (n =9)428N O S (n =16)1.2D 0C y c l o p h o s p h a m i d e +P PN o r e m i s s i o n T r a n s p l a n t e c t o m y M 24M C D D 10(n =16)M C D (n =20)C E L L (n =12)T r a n s p l a n t e c t o m y M 24C E L LM e a n ±S D 9.2±6.93.9±3.319.5±11.6D ,d a y ;M ,m o n t h ;C s A ,c y c l o s p o r i n e ;I V ,i n t r a v e n o u s ;P P ,p l a s m a p h e r e s i s ,CE L L ,c e l l u l a r ;C O L ,c o l l a p s i n g ;N O S ,m o t o t h e r w i s e s p e c i f i e d ;T I P ,t i p l e s i o n ,P H ,p e r i h i l a r ;M C D ,m i n i m a l c h a n g e d i s e a s e ;X ,r e t u r n i n d i a l y s i s .1325Variants of FSGS and risk of recurrenceTable3.Course of FSGS variants on kidney biopsies performed in transplant recipients with recurrence of proteinuria on successive kidney transplant Second allograft Third allograftBiopsy at time Biopsy at timeof recurrence Month3Biopsy Month12of recurrence Month3Month12 Native First(number of(number biopsy(number(number of(number biopsy(number Patients kidney allograft glomeruli)of glomeruli)of glomeruli)glomeruli)of glomeruli)of glomeruli) 3CELL COL MCD(n=12)NOS(n=10)NOS(n=7)MCD(n=9)MCD(n=18)MCD(n=16) 7CELL NOS MCD(n=23)MCD(n=14)X19NOS COL MCD(n=16)MCD(n=15)NOS(n=8)32PH COL MCD(n=15)MCD(n=12)TIP(n=12)36NOS PH MCD(n=9)MCD(n=20)MCD(n=14)42NOS CELL MCD(n=7)MCD(n=18)MCD(n=20)MCD(n=17)MCD(n=12)MCD(n=9) CELL,cellular;COL,collapsing;NOS,not otherwise specified;TIP,tip lesion;PH,perihilar;MCD,minimal change disease;X,return in dialysis.are on long-term permanent therapy with PP).Conversely, patients who never achieved complete and sustained re-mission developed FSGS lesions.Histological findings on native kidneys were not predictive of either recurrence or category of FSGS variant on3-month or12-month biop-sies,and only3patients(Table2:patients7,16and24) had the same variants on native kidney biopsies and on 12-month transplant biopsies.In four cases(Table2:pa-tients3,7,31and35),the FSGS variant changed during the post-transplant course.Six patients experienced a recurrence on a second allo-graft and two on a third one.Histological findings were different from native kidney on the first,second or even the third allograft(Table3).In the recurrence group,6/42patients lost their graft during the first year and12/42patients during the first5 years.The five-year graft survival in this group was71.5%. In the group exempt of recurrence,the5-year graft survival was85.5%Only one patient had an electron microscopy study(Ta-ble2:patient6)at the time of nephrotic-range proteinuria recurrence with the observation of foot process effacement. DiscussionAn early transplant biopsy in patients with recurrence of nephrotic-range proteinuria without signs of rejection shows normal glomeruli by light microscopy and no de-posit by immunofluorescence.Electron microscopy,when performed,shows widespread foot process effacement.Le-sions of FSGS develop only after several weeks as a con-sequence of persistent proteinuria.Therefore,the term of FSGS recurrence is confusing,and in this paper the re-currence was considered when massive proteinuria occurs soon after transplantation even if renal biopsies did not show histological lesion of FSGS.In the absence of glomerular immune deposits and in the absence of histological sign of rejection,the diagnosis of recurrence is almost certain. Moreover,those patients with recurrence of massive pro-teinuria who respond to therapy with complete and sus-tained remission of proteinuria do not develop FSGS as it was the case in17/42patients in our series.In native kidney,we found a distribution of FSGS vari-ants different from the adult American population,but sim-ilar to the only paediatric report available[23–25].NOS and CELL were the most frequent variants observed,with a greater representation of NOS,supporting the idea that it is the commonest variant even in a paediatric popula-tion.Interestingly,we observed TIP(7.8%)and PH(7.8%) variants in children.The fact that all FSGS variants were present in the native kidney,even the TIP lesions that are often steroid sensitive and have a more favourable outcome [15],demonstrates that all FSGS variants can lead to ESRD. Interestingly,perihilar variant,usually observed in the sec-ondary form[26],was present in native kidneys of four patients who experienced recurrence.Furthermore,after transplantation,two patients suffering from recurrence had a perihilar variant.At the time of proteinuria recurrence,MCD was the most frequently histological pattern.Only one patient(patient38) had already developed FSGS(PH variant)on Day15,on the transplant kidney.One might hypothesize that this le-sion was present in the donor kidney.But,this donor had no past history of proteinuria,and pre-transplant biopsy did not show any glomerular abnormality.Furthermore, the following course of this recipient was concordant with recurrence.Thus,we interpreted this as the result of a prob-ably uncontrolled and more aggressive disease leading to the rapid constitution of FSGS.Interestingly,early FSGS was also observed in two patients in a recent study(COL on Day7and NOS on Day10)[21].At Month3and subsequently,the incidence of MCD decreased progressively while FSGS was increasingly ob-served in those patients with persistent proteinuria.Con-versely,at1year,there was a strong association between sustained remission and the absence of FSGS.So,on se-rial biopsies,the incidence of MCD decreased over time and the incidence of FSGS variants increased.As we previously observed,patients who achieved complete remission under intensive and prolonged treatment of recurrence did not develop FSGS lesion[27].These serial biopsies indicated a correlation between the clinical status and histological findings.During this period(1984–2007),many treatments were used to treat INS recurrence with good efficiency(5-year graft survival is71.5%in our study population).Concor-dant studies had demonstrated the relative efficiency of the association of intravenous cyclosporine with plasma ex-changes to obtain and sustain complete remission[27,28].1326G.Canaud et al.Fig.3.Summary of the different patterns of FSGS variant observed on a native kidney of patients with recurrence of proteinuria in the nephrotic range,and variant observed on a transplant kidney.This series confirmed that rituximab did not dramatically change the course of recurrence,and did not consistently reach remission[29,30].The aims of this study were to determine if the Columbia classification of FSGS(1)could predict the risk of re-currence of INS,and(2)the type of FSGS variant after transplantation.Here,we showed that all FSGS variants, even the PH variant,were present in the native kidney of patients with idiopathic NS progressing to ESRD,and that no difference in their distribution was observed between recurrent and non-recurrent patients.In other words,re-currence of proteinuria/NS may be observed whatever the type of FSGS variant and conversely patients with an FSGS variant(COLL,CELL)were usually associated with a se-vere clinical course.On the other hand,all types of FSGS variants were observed in the transplant kidney and we did not find any correlation between the variant present in the native kidney and those occurring in transplanted kidney (Figure3).It should be stressed that only3/21had the same FSGS variant in native and transplanted kidneys.In-terestingly,all FSGS variants were observed in transplanted kidneys with recurrence.The discrepancy between variant native and transplant kidneys may have several explanations.Several factors may play a role in the development of FSGS in nephrotic pa-tients.First of all,the probable immunological course of the disorders may lead to the production of the pathogenic circulating factor.This factor is the same before and after transplantation,and its consequences could be modified by the immunosuppressive regimen.The long-term use of im-munosuppressive drugs,especially calcineurin inhibitors, promotes obliterative vascular lesions and ischaemic ter-ritories that can lead to the development of FSGS.The form associated with ischaemia is usually the collapsing variant[31,32].Secondly,the genetic background of the transplanted kidney,different from the recipient,may be also in question.Supporting this hypothesis,recent data have highlighted that multiple MYH9SNPs and haplotypes were recessively associated with FSGS[33].In addition,the haemodynamic condition associated with a solitary kidney could favour the development of FSGS,or more specifi-cally,the PH variant.T wo other variables render it difficult to classify the variant:firstly biopsy sampling,and sec-ondly the delay between onset of proteinuria and biopsy. In this series,biopsy specimens were serially studied and a mean of13.7glomeruli per biopsy was examined allowing a precise evaluation of FSGS variants.Indeed,late biopsies after the beginning of the disease are more difficult to in-terpret because all FSGS variants found at early stages of the disease might progress towards NOS variant[15].Following transplantation,the rate of recurrence of pro-teinuria varies between30%and50%and is associated with a poor graft outcome[8–14].Several risk factors for recurrence have been identified including older age at on-set on disease,rapid progression to ESRD and recurrence on a previous graft but none of them can clearly distin-guish between patients who will and those who will not be affected by recurrence[34].On native kidneys,the recent Columbia University classification is interesting since it combines epidemiological data on native kidneys with in-formation on the prognosis and response to treatment.The two main variants are NOS and CELL.The COL variant is associated with the worse prognosis i.e.poor or no response to treatment.In contrast,patients with the TIP variant of-ten respond to corticosteroids and rarely progress to ESRD. NOS,PH and CELL variants are associated with an inter-mediate prognosis[23,25,35].The aim of our study was to evaluate the potential value of the Columbia classification of FSGS to predict risk and the type of recurrence,ques-tions that had never been addressed.Ijpelaar et al.recently identified in21cases three distinct patterns of recurrence of FSGS in renal transplant:firstly,recurrence of the same variant of FSGS;secondly,recurrence of the same variant of FSGS after an intermediate state of MCD and a third pattern of recurrence with a different FSGS variant in the allograft[21].The authors found a high rate of concordance of FSGS variant between native and transplanted kidneys. The discrepancy with our results may be explained by sev-eral differences.On the native kidney,diagnosis of FSGS and the type of variant in our series were all performed on renal biopsies with a mean of13.7±4.6(range6–24) glomeruli,whereas it was performed on the nephrectomy specimen in6/19native kidney in the Ijpelaar study.In-deed,the predominant pattern of NOS variant observed in their study is certainly explained by the course of disease progression as it was suggested in the Columbia Classifica-tion[15].In the same way,after kidney transplantation,in the Ijpelaar study,transplant biopsies were performed late, at a mean of783.4days(range10–2555),suggesting that most biopsies at the time of recurrence,which occurred classically early after transplantation,were not available.In our series,renal biopsies were performed within the first3 months of recurrence,an explanation for the diverse type of FSGS lesion observed.In conclusion,our series is the first to report serial biopsy data in kidney transplant recipients with recurrent nephrotic syndrome.Our results suggest that the Columbia classifi-cation of FSGS is not helpful in predicting neither recur-rence nor the histological variant of FSGS when recurrence develops.Conflict of interest statement.None declared.References1.Barisoni L,Schnaper HW,Kopp JB.A proposed taxonomy for thepodocytopathies:a reassessment of the primary nephrotic diseases.Clin J Am Soc Nephrol2007;2:529–5422.Habib R,Levy M,Gubler MC.Clinicopathologic correlations in thenephrotic syndrome.Paediatrician1979;8:325–3481327Variants of FSGS and risk of recurrence。
Twist和E-cadherin蛋白在口腔鳞状细胞癌中的表达及临床意义
• 1884 •吴和刚，等 Twist和E-cadherm蛋白在口腔鳞状细胞癌中的表达及临床意义❖头领肿瘤❖Twist和E- cadherin蛋白在口腔鱗状细胞癌中的表达及临床意义吴和刚，张续，李天永，袁森辅，郭宇，朱坤婷Expression of Twist and E-cadherin in oral squamous cell carcinoma and its clinical significanceWu Hegang,Zhang X u,L i Tianyong,Yuan Senfu,Guo Yu,Zhu KuntingDepartment o f Pathology y the First Peoples Hospital in Yibin, Sichuan Yibin 644QQQ, China.【Abstract 】Objective: To investigate the expression o f tra n scrip tio n factor T w ist and e p ith e lia l c e ll ca lciu m m u cin(E - c a d h e rin) in oral squamous c e ll c a rcin o m a( O SC C) and to explore th e ir re la tio n sh ip between w ith the o ccu rrence and developm ent o f OSCC. Methods ：Im m uno histoche m istry SP m ethod was used to detected the expression o fT w ist and E - cad herin in20cases o f norm al oral mucosa tissues and 78 cases o f OSCC tissues. C orrelation betweenexpression o f T w ist and E - cad herin in OSCC was analysed by Spearman ra nk correlatio n analysis. Results：The expression o f T w ist in OSCC tissues was s ig n ific a n tly h ig h e r than norm al oral mucosa tissues ( P< 0. 05 ) . The expressiono f E - cad herin in OSCC tissues was s ig n ific a n tly low er than norm al oral mucosa tissue s( P< 0. 05 ) . The expression o fE - ca d herin in OSCC tissues o f stage IH and IV was low er than stage I and II (P< 0. 05 ).The expression o f E -cad herin in OSCC tissues o f patients w ith lym p h node metastasis was low er than th a t non m etastasis( P< 0. 05 ) . Theexpression o f T w ist in OSCC tissues o f stage IH and IV was h ig h e r than stage I and II (P< 0. 05 ).The expression o fT w ist in OSCC tissues o f patients w ith lym p h node metastasis was high er than that non metastasis (P<0.05).The expression o f T w ist in OSCC was negatively correlated w ith the expression o f E -c a d h e rin(r = -0.639,P< 0. 05 ).Conclusion：The abnorm al expression o f T w ist and E - cad herin in OSCC tissu e s,a n d th e ir expression was h ig h ly corre la te d, suggesting that they may pa rticip a te in the occurrence and developm ent o f O SC C, invasio n and metastasis.Com bined detection o f T w ist and E -cad herin has great c lin ic a l values in the p re v e n tio n, d iag nosis, treatm ent andprognosis o f OSCC.[Key words】oral squamous c e ll c a rcin o m a，T w is t，E - c a d h e rin，im m unohistochem istryM o dem Oncology 2017,25(12)：1884 -1887【摘要】目的:研究转录因子T w is t和上皮细胞钙黏蛋白（E - c a d h e rin)在口腔鳞状细胞癌（O S C C)中的表达，探讨其与O S C C发生发展的关系。
宫内发育迟缓大鼠胰腺骨保护素的表达及其对胰腺发育的影响
·716·宫内发育迟缓 （intrauterine growth retardation，IUGR ） 是指胎儿未能达到其遗传基因影响下应有的大小。
IUGR可增加成年期发生胰岛素抵抗、2型糖尿病等代谢综合征的风险［1-3］，其机制尚不十分清楚。
目前普遍被认可的机制是“节俭表型假说”［2］，该假说指出胎儿及新生儿早期营养不良会导致胰岛β细胞的发育异常及胰岛功能损伤，这种解剖结构和功能的变化可能导致胰岛素敏感性降低，进而成年后发生2型糖尿病。
骨保护素 （osteoprotegerin，OPG ） 是肿瘤坏死因子受体家族的成员，1997年由SIMONET等［4］发现，因具有保护骨的作用而得名。
近年的研究［5-8］发现，OPG在人类及大鼠胰岛中均有表达。
RIECK等［5］通过芯片筛查发现OPG在怀孕、肥胖和β细胞损伤动物模型中的表达水平均升高，提示OPG与胰岛β细胞之间存在密切的关系。
但OPG对胰岛β细胞的具体影响目前仍存在争议［6-8］。
· 论著 ·宫内发育迟缓大鼠胰腺骨保护素的表达及其对胰腺发育的影响唐诗，辛颖 （中国医科大学附属盛京医院小儿内分泌遗传代谢科，沈阳 110004） 摘要 目的 探讨宫内发育迟缓 （IUGR ） 大鼠胰腺组织中骨保护素 （OPG ） 的表达水平及其对胰腺发育的影响。
方法 在生后1 d 及1、3、12周测量IUGR 组和对照组 （CON 组） 大鼠体质量、胰腺质量、空腹血糖及胰岛素水平，HE 染色观察胰岛形态，免疫荧光双染观察胰岛β细胞增殖情况，Westen blotting 及实时 PCR 检测胰腺中OPG 蛋白和mRNA 的表达。
结果 与CON 组相比，IUGR 组大鼠胰岛体积小，β细胞增殖水平低，且在12周时出现胰岛素抵抗。
IUGR 组各时间点胰腺组织中OPG 蛋白及mRNA 表达水平均明显低于CON 组。
结论 IUGR 大鼠胰腺中OPG 蛋白及mRNA 表达减少，可能导致其胰腺发育落后，胰岛β细胞增殖水平下降。
百令胶囊与斯奇康联合治疗慢阻肺缓解期的临床分析
China &Foreign Medical Treatment2017 NO.5药物与临床DO I:10.16662/ki.l674-0742.2017.05.114百令胶囊与斯奇康联合治疗慢阻肺缓解期的临床分析[摘要]目的探讨百令胶囊与斯奇康联合治疗慢阻肺(COPD )缓解期的临床效果及安全性。
方法便利选择2012年7 月一2013年7月期间该院收治的50例慢阻肺缓解期患者作为研究对象，并随机分为观察组和对照组，每组25例， 观察组给予百令胶囊与斯奇康联合治疗，对照组给予单一百令胶囊治疗。
观察两组患者的急性发作次数、住院次数、 死亡人数及肺功能下降指标。
结果观察组3年期间内的急性发作人数（8、10、11例）、平均天数（11.2±2.0)、（8.6±1.9)、 (8.9±2.3)d ，发作后的平均住院天数（8.3±1.3)、（7.3±1.6)、（5.3±0.9)d 等均明显优于对照组，组间差异有统计学意义 (P <0.05);观察组治疗后的肺功能指标[FVC (1.6±0.2)V /L 、FEV 1(61.3±0.9)%c 、FEV 1/FVC (63.6±0.7)%c ]显著优于对照 组，组间差异有统计学意义(P <0.05)。
结论百令胶囊与斯奇康联合治疗COPD 的临床疗效显著，能够显著提髙患者 的抵抗能力，预防呼吸道感染引起的慢阻肺急性发作，提髙患者的生存质量。
[关键词]慢阻肺;缓解期;百令胶囊;斯奇康;干预治疗[中图分类号]R 563.9[文献标识码]A[文章编号]1674-0742(2017)02(b )-0114-03Clinical Analysis of Corbrin Capsule and Siqikang in Treatment of ChronicObstructive at Remittent StageH UANG X iu -fe nDepartment of Respiratory Medicine , Puer People,s Hospital , Puer , Yunnan Province , 665000 China[Abstract] Objective To discuss the clinical effect and safety of corbrin capsule and siqikang in treatment of chronic ob structive at remittent stage . Methods Convenient selection 50 cases of patients with chronic obstructive at remittent stage admitted and treated in our hospital from July 2012 to July 2013 were selected as the research objects and randomly divid ed into two groups with 25 cases in each , the observation group were treated with corbrin capsule and siqikang , while the control group were treated with corbrin capsule , and the acute attack frequency , hospitalization time , death number and lung function decrease indexes of the two groups were observed . Results The acute attack number ( 8 cases , 10 cases and 11 cases ), average time (11.2±2.0)，(8.6±1.9)，(8.9±2.3)d and the average length of stay after attack (8.3±1.3)，(7.3±1.6)，(5.3± 0.9)d in the observation group within 3 years were obviously better than those in the control group , and the differences had statistical significance (P <0.05), after treatment , the lung function indexes [FVC (1.6±0.2)V /L ,FEV 1(61.3±0.9)%,FEV 1/ FVC (63.6±0.7)%]in the observation group were obviously better than those in the control group , and the differences be tween groups had statistical significance (P <0.05). Conclusion The clinical curative effect of corbrin capsule and siqikang in treatment of COPD is obvious , which can obviously improve the resistance ability of patients , prevent the acute attack of chronic obstructive caused by the respiratory tract infection and improve the quality of life of patients .[Key words] Chronic obstructive ; Remittent stage ; Corbrin capsule ; Siqikang ; Intervention treatment慢性阻塞性肺病（COPD )严重影响患者的生活质量，而呼吸道感染是导致COPD 加重的主要原因，如何[作者简介]黄秀芬（1966.11-)，女，哈尼族，云南普洱人，本减少慢阻肺的发作次数和延缓病程进展，成为了 COPD 黄秀芬普洱市人民医院呼吸内科，云南普洱665000科，副主任医师，主要从事呼吸内科临床工作缓解期治疗的关键[1]。
Prucalopride通过促进自噬抑制胶质瘤细胞U251增殖
• 1340 •霍会永，等 Prucalopride通过促进自噬抑制胶质瘤细胞U251增殖184. RNAi screening[ J]. Cell Biosci ,2015 ,5 ：66.[17] Wang H,L iu K,Fang B A M,e t al. Identification of acetyltrans- (编校：谈静）ferase genes ( HAT1 and KAT8 ) regulating HBV replication byPrncalopride通过促进自嗤抑制胶质瘤细胞U251增殖霍会永1刘冰1，曹凌1赵现1曹妍1，薛靖1王如科2,李军涛1Prucalopride inhibits cell proliferation of glioblastoma cell U251 by promoting autophagyHuo Huiyong1,Liu Bing1 ,Cao Ling1,Zhao Xian1,Cao Yan1,X ue Jing1, Wang Ruke2 ,Li1Department of Second Neurology；2Department of Neurosurgery,Handan Central Hospital,Hebei Handan056000, China.【A b stract】O bjective:To investigate the effect of Prucalopride on the proliferation，migration，autophagy，and apoptosis of glioblastoma cell U251 and to explore the related signaling pathways.M ethods:CCK8 assay，Flowcytometry，Trans'well chamber and Western blot were used to detect cell proliferation，migration，aut sion of AKT/mTOR signaling related proteins,respectively.R esu lts：Prucalopride inhibited cell proliferation and migration of U251cells(P< 0.05 ).Prucalopride induced apoptosis of U251cells(P< 0.05 ).Prucaloprid increased theexpression level of B a and Active Caspase3,decreased expression of Bcl-2. Prucalopride treatment also increasedexpression of autophag- related proteins(LC3 II/I ,Beclin1) ,reduced the level of p62 in U251 c e ll(P<0. 05).C onclusion:Prucalopride can inhibit cell proliferation，migration.Induce autophagy and apoptosis，which is achievedby inhibiting activation of the AKT/mTOR signaling pathway in U251 cell.【K ey w ord s】Prucalopride，glioma，autophagy，AKT/mTORsignalingModern Oncology2018,26(09) ：1340 - 1344【摘要】目的:研究P r u c lo p r d e对胶质瘤U251细胞增殖、自噬、凋亡的影响，并探讨其相关信号通路。
蛋白酶激活受体-2在特发性肺纤维化
Role of Protease-activated Receptor-2in Idiopathic Pulmonary FibrosisMalgorzata Wygrecka1,Grazyna Kwapiszewska2,Ewa Jablonska1,Susanne von Gerlach3,Ingrid Henneke2,Dariusz Zakrzewicz1,Andreas Guenther2,Klaus T.Preissner1,and Philipp Markart21Department of Biochemistry,2Department of Internal Medicine,and3Department of Pathology,Faculty of Medicine,University of Giessen Lung Center,Giessen,GermanyRationale:Activation of the coagulation cascade has been demon-strated in pulmonaryfibrosis.In addition to its procoagulant func-tion,various coagulation proteases exhibit cellular effects that mayalso contribute tofibrotic processes in the lung.Objective:To investigate the importance of protease-activated re-ceptor(PAR)-2and its activators,coagulation factor VIIa(FVIIa)/tissue factor(TF),in the development of idiopathic pulmonaryfibrosis(IPF).Methods:Expression and localization of PAR-2and its activators wereexamined in IPF lung tissue.The ability of PAR-2to mediate variouscellular processes was studied in vitro.Measurements and Main Results:Expression of PAR-2was stronglyelevated in IPF lungs and was attributable to alveolar type II cells andfibroblasts/myofibroblasts.Transforming growth factor-b1,a keyprofibrotic cytokine,considerably enhanced PAR-2expression inhuman lungfibroblasts.FVIIa stimulated proliferation of humanlungfibroblasts and extracellular matrix production in a PAR-2–dependent manner,but did not initiate differentiation offibroblastsinto myofibroblasts.PAR-2/FVIIa-driven mitogenic activities weremediated via the p44/42mitogen-activated protein kinase pathway and were independent of factor Xa and thrombin production. Proproliferative properties of FVIIa were markedly potentiated in the presence of TF and abrogated by TF antisense oligonucleotides. Hyperplastic alveolar type II cells overlyingfibroblastic foci were found to be the source of FVII in IPF lungs.Moreover,TF colocalized with PAR-2onfibroblasts/myofibroblasts in IPF lungs. Conclusions:The PAR-2/TF/FVIIa axis may contribute to the devel-opment of pulmonaryfibrosis;thus,interference with this pathway confers novel therapeutic potential for the treatment of IPF. Keywords:protease activated receptor-2;idiopathic pulmonaryfibrosis; coagulation factor VII;tissue factorIdiopathic pulmonaryfibrosis(IPF)represents a specific form of chronicfibrosing idiopathic interstitial pneumonia characterized by the histological appearance of a usual interstitial pneumonia (UIP)pattern(1).Distinctive features of IPF are injury and activation of epithelial cells,subepithelial formation offibro-blast foci,and excessive deposition of extracellular matrix (ECM)proteins,such as collagen,fibronectin,or osteopontin (2–5).IPF is typically characterized by a progressive and usually fatal course with a median survival of2to3years(6).Accumulating evidence suggests that activation of the co-agulation cascade may play a role in the pathogenesis of pulmonaryfibrosis.Increased procoagulant activity has been observed in the bronchoalveolar lavagefluids(BALFs)of patients with IPF.The procoagulant activity observed under these conditions arises from the imbalance between locally produced pro-and anticoagulant factors,in combination with leakage of plasma proteins into the alveolar space.Tissue factor (TF)in association with factor VIIa(FVIIa)and inhibition of urokinase by plasminogen activator inhibitor-1are major fac-tors that are responsible for the intraalveolar accumulation of fibrin(7,8).Fibrin deposits have been found in lung biopsies from patients with pulmonaryfibrosis,and they have been associated with the development offibrotic lesions(9).Besides their important role infibrin formation,it is now well recognized that coagulation proteases exert profibrotic cellular effects via activation of protease-activated receptors (PARs).At present,four PARs have been described:PAR-1to PAR-4.Thrombin activates PAR-1,PAR-3,and PAR-4, whereas factor Xa(FXa)cleaves either PAR-1or PAR-2, depending on cell type and cofactor expression.PAR-2is also activated by trypsin,tryptase,neutrophil proteinase-3,TF/ FVIIa,and TF/FVII/FXa complexes(10,11).To date,some studies have investigated the contribution of PAR-1to the development of pulmonaryfibrosis.Activation of PAR-1by FXa or thrombin was found to promotefibroblast proliferation, procollagen production,cytokine release,andfibroblast-to-myofibroblast differentiation(12–14).A potential role of PAR-1infibrotic processes was further underscored by the finding that PAR-1–deficient mice are protected from bleomy-cin-induced lungfibrosis(15).In line with this observation, direct inhibition of FXa or thrombin attenuated thefibrotic response in the bleomycin model as well(14,16).(Received in original form September15,2010;accepted infinal form March10,2011) Supported by the German Research Foundation(DFG,KFO118),the Excellence Cluster‘‘Cardiopulmonary System’’(ECCPS),the University Medical Center Giessen and Marburg(UKGM),the European Commission through FP7(Euro-pean IPF Network),and the German Ministry of Education and Research(German Network for Diffuse Parenchymal Lung Diseases).Contribution of the authors:Conception and design:M.W.,A.G.,P.M.;analysis and interpretation:M.W.,G.K.,E.J.,S.v.G.,I.H.,D.Z.,K.T.P.,P.M.;drafting the manuscript for important intellectual content:M.W.,A.G.,P.M. Correspondence and requests for reprints should be addressed to Malgorzata Wygrecka,Ph.D.,Department of Biochemistry,Faculty of Medicine,University of Giessen Lung Center,Friedrichstrasse24,35392Giessen,Germany.E-mail: malgorzata.wygrecka@innere.med.uni-giessen.deThis article has an online supplement,which is available from this issue’s table of contents at Am J Respir Crit Care Med Vol183.pp1703–1714,2011Originally Published in Press as DOI:10.1164/rccm.201009-1479OC on March11,2011 Internet address:Although the role of PAR-1in the development of lung fibrosis has been documented,much less is known about the contribution of PAR-2to this pathological condition.PAR-2 has been suggested to play a role in tissue repair processes in the liver,pancreas,and kidney(17–19).Therefore,it is tempting to hypothesize that PAR-2may play a role in thefibroprolifer-ative process occurring in the lungs of patients with IPF.To explore this idea,the expression and activity of the PAR-2/TF/ FVII axis was investigated in IPF lungs,with particular atten-tion being paid to PAR-2–driven lungfibroblast activation,the major hallmark offibrotic processes.Some of the results of these studies have been previously reported in the form of an abstract(20).METHODSA detailed description of routine methodologies is provided in the online supplement.Only nonstandard procedures and specialized materials are described in this section.Study PopulationThe investigations have been conducted according to Declaration of Helsinki principles and were approved by the local institutional review board and ethics rmed consent was obtained from either the patients or their next-of-kin.BALF was obtained byflexible fiberoptic bronchoscopy from20spontaneously breathing healthy volunteers and from40spontaneously breathing patients with IPF. Diagnosis of IPF was settled on the basis of the American–European Consensus Criteria(1).In20patients,diagnosis was confirmed by surgical lung biopsy and forwarded an UIP pattern in every case.In addition,lung tissue was obtained from24patients with IPF who underwent lung transplantation.IPF diagnosis was based on both clinical criteria as well as proof of an UIP pattern.Unused donor lungs served as a control(n510).Table1shows the demographic and clinical characteristics of the patient cohort.Cell Isolation and StimulationFibroblasts and alveolar type II cells were isolated from donor and IPF lungs as described(21,22).Fibroblasts were stimulated for various times with FXa(50nM),FVIIa(50nM),thrombin(65U/L), inactivated FVIIa(FVIIi;50nM)(all from American Diagnostica, Greenwich,CT);peptide agonist for PAR-2(AP;2-furoyl-LIGRLO-NH2,100m M),scrambled peptide control(RP;trans-cinnamoyl-OLIGRL-NH2,100m M)(both kindly provided by A.Meinhardt, Department of Anatomy and Cell Biology,Justus Liebig University, Giessen,Germany);or transforming growth factor(TGF)-b1(10ng/ml; R&D Systems,Wiesbaden,Germany).In some experiments,cells were transfected with small interfering RNA(siRNA)sequences directed against human PAR-2(Santa Cruz Biotechnology,Santa Cruz,CA)or TF(Ambion,Austin,TX)or with vectors containing full-length cDNA for human PAR-2or TF.Cell Proliferation AssayProliferation of human lungfibroblasts(HLFs)was determined by a DNA synthesis assay based on the uptake of[3H]thymidine. StatisticsStatistical analyses were performed in R version2.3.1.Deviations from the normal distribution were tested by Shapiro-Wilk test.All in vitro data were normally distributed and therefore these data are presented as means(6SD).Clinical data are given as medians and interquartile range.The box-and-whisker plots indicate the median andfirst and third quartiles;the whiskers are extended to the most extreme value inside the1.5-fold interquartile range.Differences between two groups were tested by Student t test and Wilcoxon rank sum test according to the distribution of the data.All tests were performed with an un-directed hypothesis(two-sided).The level of statistical significance was set at5%.RESULTSPAR-2Expression Is Elevated in IPF LungsInitially,we analyzed the expression of PAR-2in IPF lungs. Elevated PAR-2mRNA(Figure1A)and protein(Figures1B and1C)levels were observed in the lung homogenates of patients with IPF.Immunohistochemical staining of donor lung sections demonstrated immunoreactivity for PAR-2in alveolar macrophages and alveolar type II(ATII)cells(Figure1D), whereas in IPF lungs strong PAR-2staining was associated with hyperplastic ATII cells andfibroblasts/myofibroblasts(Figures 1D and1E).Western blot analysis revealed enhanced pro-duction of PAR-2infibroblasts isolated from IPF lungs as compared withfibroblasts isolated from donor lungs(Figures1F and1G).Immunofluorescence analysis confirmed these results (Figure1H,top).Furthermore,fibroblasts derived from IPF lungs showed a marked increase in the number of a-smooth muscle actin(a-SMA)–positive cells,indicating their myofibro-blast phenotype(Figure1H,bottom).Asfibroblasts are key effector cells in the development offibrosis,in further studies we focused on the role of PAR-2in the regulation of various processes in this cell population.TGF-b1Up-Regulates PAR-2mRNA and Protein Levels in HLFs To investigate how PAR-2expression may be regulated in pulmonaryfibrosis,donor HLFs were stimulated with the key profibrotic growth factor TGF-b1,and PAR-2production was determined by quantitative real-time polymerase chain reaction (qRT-PCR)and Western blotting.TGF-b1increased PAR-2 mRNA and protein expression,with maximal effects observed at20and72hours of treatment,respectively(Figures2A–2C). Similar results were obtained when HLFs were treated with other profibrotic cytokines,such as platelet-derived growth factor-BB or insulin-like growth factor-1(data not shown). FXa and FVIIa Induce Proliferation of HLFsWe next investigated the induction of donor HLF proliferation by PAR-2activators such as FXa and FVIIa.Both FXa and FVIIa increased lungfibroblast proliferation as assessed by cell counting(Figure3A).Elevated HLF proliferation was also evident by enhanced[3H]thymidine incorporation(Figure3B) and increased immunostaining for Ki67(Figures3C and3D).In accordance with these data,a time-dependent rise in the expression of cyclin D1on stimulation of HLFs with FXa or FVIIa was observed(see Figure E1in the online supplement). Of note,mitogenic effects induced by FXa and FVIIa were as pronounced as those induced by thrombin(Figures3A23D). Neither the thrombin inhibitor hirudin nor the FXa inhib-itor TENSTOP blocked FVIIa-induced proliferation of HLFsTABLE1.DEMOGRAPHIC CHARACTERISTICS AND CLINICALDATA OF PATIENT COHORTVariable IPF(BALF)IPF(lung tissue)Subjects,n4024Age(yr),mean6SD60.3612.156.3618.7Sex(male/female),n/n30/1019/5Smoking status(never/former/current),n/n/n20/15/510/14/0FVC%predicted,mean6SD65.2625.057.4619.5D L CO%predicted,mean6SD45.6613.829.1618.0Histological confirmation of a UIP pattern,%50100Definition of abbreviations:BALF5bronchoalveolar lavagefluid;D L CO5diffusion capacity of the lung for carbon monoxide;IPF5idiopathic pulmonaryfibrosis;UIP5usual interstitial pneumonia.1704AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL1832011(Figure 3E).This indicates that FVIIa-dependent mitogenic effects are not a result of thrombin and FXa generation,which are the downstream products of FVIIa activation.FVIIa-induced Proliferation of HLFs Requires PAR-2and TFTo determine the potential role of PAR-2in FXa-and FVIIa-induced cell proliferation,donor HLFs were transfected with PAR-2–and/or TF-specific siRNAs and then stimulated with the respective protease.Transfection of HLFs with PAR-2(siPAR-2)or TF (siTF)siRNA caused significant knockdown of these proteins as demonstrated by RT-PCR and Western blotting (Figure 4A).Moreover,PAR-2siRNA did not affect ex-pression of PAR-1,indicating target gene specificity (Figure E2).As shown in Figure 4B,knockdown of PAR-2abolished the mitogenic response of HLFs to FVIIa but did not influence the proliferation rate induced by FXa or thrombin.Depletion of TF almost completely inhibited the mitogenic response of HLFs to FVIIa but did not affect HLF proliferation on stimulation with FXa or thrombin (Figure 4B).Similar results were obtained when the cells were simultaneously transfected with siRNAs specific for PAR-2and TF.To further confirm a crucial role of PAR-2and TF in FVIIa-induced HLF proliferation,we evaluated the effect of PAR-2and/or TF overexpression on this process.Transfection of PAR-2(pPAR-2)or TF (pTF)vectors into HLFs considerably in-creased expression of these proteins as assessed byWesternFigure 1.Expression of protease-activated receptor (PAR)-2is elevated in idiopathic pulmo-nary fibrosis (IPF)lungs.(A )PAR-2expression in lung ho-mogenates of donors (n 510)and patients with IPF (n 524)as assessed by quantitative real-time polymerase chain re-action (qRT-PCR).qRT-PCR re-sults are expressed as D C t (difference in cycle threshold [C t ]between the housekeeping gene [PBGD;porphobilinogen deaminase]and the gene of interest).Significance level is indicated.(B )PAR-2expression in lung homogenates of donors and patients with IPF as assessed by Western blotting.The specificity of the detected band was proven by preincu-bating the anti–PAR-2antibody (a PAR-2)with its blocking pep-tide (BP).Representative do-nors (4of 10)and patients (9of 24)are shown.(C )Densito-metric analysis of (B ).Signifi-cance level is indicated.(D )Representative serial lung sec-tions from patients with IPF and donors,stained for PAR-2,a -smooth muscle actin (a -SMA),or pro–surfactant protein C (proSP-C).Negative controls,including no anti–PAR-2anti-body,isotype control,and anti–PAR-2antibody preab-sorbed with blocking peptide,are demonstrated.Arrowheads indicate alveolar type II (ATII)cells,asterisks represent alveo-lar macrophages,and arrows indicate fibroblasts/myofibro-blasts.Original magnification,320.Scale bar :100m m.(E )Double immunostaining of IPFlung sections for PAR-2and a -SMA.Arrows indicate PAR-2–and a -SMA–positive fibroblasts/myofibroblasts.Insets show the negative control.Original magnification,343.Scale bar :20m m.(F )PAR-2expression in human lung fibroblasts (HLFs)isolated from donor (n 510)and IPF (n 524)lungs.The specificity of the detected band was proven by preincubating anti–PAR-2antibody (a PAR-2)with its blocking peptide (BP).Representative donors (4of 10)and patients (4of 24)are shown.(G )Densitometric analysis of (F ).Significance level is indicated.(H )Donor and IPF lung fibroblasts cultured in Dulbecco’s modified Eagle’s medium supplemented with 10%fetal calf serum and 1%penicillin–streptomycin were stained for PAR-2(top )and a -SMA (bottom ).Insets show the negative control.Original magnification,340.Scale bar :10m m.Wygrecka,Kwapiszewska,Jablonska,et al.:PAR-2in IPF 1705blotting (Figure 4C).Overexpression of PAR-2and TF alone partially enhanced cell proliferation on treatment with FVIIa.This effect was strongly potentiated when PAR-2and TF were overexpressed simultaneously (Figure 4D).Of note,overex-pression of PAR-2and TF alone or together did not induce any changes in HLF proliferation in response to FXa or thrombin (Figure 4D).Collectively,these data strongly suggest that PAR-2and TF are required for FVIIa-triggered HLF proliferation but are dispensable for the induction of HLF proliferation by FXa or thrombin.To determine the role of FVIIa catalytic activity in the induction of donor HLF proliferation,cells were incubated with FVIIi.FVIIi did not exert any proproliferative effects on HLFs,indicating the requirement of FVII proteolytic activity for the induction of HLF proliferation (Figure 4E).Two-furoyl-LIGRLO-NH 2,a potent and selective PAR-2agonist peptide (AP [23]),served as positive control.As PAR-2expression was strongly up-regulated in diseased lung fibroblasts,we sought to study the proliferation rate of donor and IPF fibroblasts in response to FVIIa.Under basal conditions no difference in growth rate between donor and IPF fibroblasts was noted;however,on FVII stimulation the mag-nitude of the mitogenic response was greater in fibroblasts isolated from the lungs of patients with IPF (Figure 4F).FVIIa Stimulates HLF Proliferation in a PAR-2/TF–dependent Manner via p44/42Signaling PathwayIntracellular mitogen-activated protein kinase (MAPK)signaling pathways play an important role in cell proliferation.Therefore,we investigated the activation of various MAPKs and Akt in response to FVIIa stimulation.FVIIa induced rapid phosphory-lation of p44/42with maximal response within 15–30minutes.In contrast,no activation of p38,c-Jun N-terminal kinase (JNK),and Akt was observed (Figure 5A).To examine whether FVIIa-stimulated phosphorylation of p44/42is PAR-2/TF dependent,we transfected donor HLFs with PAR-2–and TF-specific siRNAs before incubation with FVIIa.Knockdown of PAR-2and TF resulted in significant inhibition of FVIIa-induced p44/42acti-vation (Figure 5B).No p44/42phosphorylation was observed when the cells were incubated with FVIIi,indicating the re-quirement of FVII catalytic activity for induction of the intracellular signaling pathway (Figure 5C).Moreover,FVIIa-stimulated proliferation of donor HLFs was significantly inhibited in the presence of the selective inhibitors of MAPK/ERKkinase-1,2Figure 1.(Continued).Figure 2.Transforming growth factor (TGF)-b 1up-regulates protease-activated receptor (PAR)-2expression in human lung fibroblasts (HLFs).(A and B )Time course of PAR-2expression in donor HLFs after TGF-b 1(10ng/ml)stimulation as assessed by (A )quantitative real-time poly-merase chain reaction (qRT-PCR)and (B )Western blotting.qRT-PCR results are expressed as D C t (difference in cycle threshold [C t ]between the housekeeping gene [PBGD;porphobilinogen deaminase]and the gene of interest).Data are shown as means 6SD,n 54.*P ,0.05.The Western blot illustrated is from one representative experiment of four.(C )Densitometric analysis of (B ).Data are presented as means 6SD;n 54.*P ,0.05;**P ,0.01.1706AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 1832011(MEK1/2),PD98059and U0126(Figure 5D).Together,these data indicate that FVIIa stimulates HLF proliferation via the p44/42signaling pathway in a PAR/2-TF–dependent manner.FVIIa Does Not Induce Differentiation of Fibroblasts into MyofibroblastsDifferentiation of fibroblasts into myofibroblasts is central to the pathogenesis of pulmonary fibrosis,and therefore we examined whether FVIIa promotes this phenotypic change.As assessed by qRT-PCR and Western blotting,exposure of donor HLFs to FVIIa did not induce expression of the myofibroblast marker a -SMA (Figures 6A–6C).In contrast,a strong stimulation of a -SMA synthesis was observed in response to TGF-b 1(Figures 6A–6C).Similar results were obtained by immunofluorescence staining (Figure 6D).FVIIa Stimulates Osteopontin and Fibronectin Production in HLFExcessive deposition of ECM proteins in the lung is a hallmark of IPF,and therefore we examined whether FVII stimulates production of ECM proteins,such as fibronectin (FN),osteo-pontin (OPN),and collagen I.Exposure of donor HLFs to FVIIa stimulated the synthesis of OPN and FN in a time-dependent manner.qRT-PCR analysis demonstrated the stron-gest induction of OPN and FN mRNA expression at 20hours of treatment (Figure 7A).Maximal OPN and FN protein levels were achieved within a 72-hour stimulation period (Figures 7B and 7C).Similar results were obtained when donor HLFs were treated for 72hours with the PAR-2agonist peptide (AP),2-furoyl-LIGRLO-NH 2(Figures 7D and 7E).To examine whether FVIIa stimulated OPN and FN synthesis in a PAR-2/TF–dependent manner,we transfected HLFs with PAR-2–and TF-specific siRNAs.As shown in Figures 7F and 7G,knock-down of PAR-2and TF resulted in significant inhibition of OPN and FN expression after FVIIa stimulation.We further evalu-ated the extent of OPN and FN synthesis during FVIIa treatment in donor and diseased fibroblasts.IPF fibroblasts exhibited moderately higher OPN and FN expression at base-line in comparison with donor fibroblasts.Interestingly,on FVII stimulation OPN and FN production was greatly augmented in fibroblasts isolated from IPF lungs (Figures 7H and 7I).Of note,stimulation of donor and IPF fibroblasts with FVIIa did not have any effect on collagen I production (data not shown).FVII Is Expressed in Alveolar Type II Cells in IPF LungsAs expression of PAR-2was found to be elevated in the lungs of patients with IPF and as FVII was identified as an important PAR-2activator stimulating fibroblast proliferation and ECM production,we next sought to examine FVII antigen level and its activity in IPF bronchoalveolar lavage fluid (BALF).In-creased FVII antigen level and activity were detected in BALF of patients with IPF (Figures E3A and E3B,and Reference 8).Immunohistochemical analysis revealed no FVII-positivestain-Figure 3.Factor Xa (FXa),FVIIa,and thrombin stimulate proliferation of human lung fibroblasts (HLFs).(A )Donor HLFs were stimulated for 24hours with FXa (50nM),FVIIa (50nM),or thrombin (65U/L).Cell proliferation was assessed by cell counting.Data are presented as means 6SD from four independent experiments.**P ,0.01;***P ,0.001.(B )[3H]thymidine incorporation in HLFs treated for 24hours with FXa (50nM),FVIIa (50nM),or thrombin (65U/L).The results are expressed as relative proliferation,compared with unstimulated cells.Data represent mean values 6SD from four independent experiments,each performed in triplicate.**P ,0.01;***P ,0.001.(C )Proliferation of HLFs stimulated for 24hours with FXa (50nM),FVIIa (50nM),or thrombin (65U/L)as assessed by immunostaining for the proliferation marker Ki67.Original magnification,320.Scale bar :10m m.DAPI 549,6-diamidino-2-phenylindole.(D )Quantification of Ki67-positive cells.The number of Ki67-positive cells was determined by counting four randomly chosen fields per slide from four independent experiments.Data are shown as means 6SD.**P ,0.01.(E )HLFs were stimulated for 24hours with FXa (50nM),FVIIa (50nM),or thrombin (65U/L)in the absence or presence of hirudin (1,000U/L)or TENSTOP (0.5m M).Cell proliferation was assessed by [3H]thymidine incorporation.The results are expressed as relative proliferation compared with unstimulated vehicle-treated cells.Data represent mean values 6SD from four independent experiments,each performed in triplicate.**P ,0.01;***P ,0.001;NS 5not significant.For all proliferation assays,cells were seeded in 48-well plates,growth arrested in serum-free Dulbecco’s modified Eagle’s medium,and stimulated for 24hours in serum-free medium with respective reagents.Wygrecka,Kwapiszewska,Jablonska,et al.:PAR-2in IPF 1707ing in donor lung tissue sections,whereas a strong positive signal for FVII was observed in hyperplastic ATII cells over-lyingfibroblast foci in IPF lungs(Figure8A).Moreover,an elevated FVIIa mRNA level was detected by qRT-PCR in ATII cells isolated from the lungs of patients with IPF(Figure8B). This suggests that FVII,which is produced by hyperplastic ATII cells,may act in an auto-and paracrine fashion to regulate cellular activities in IPF lungs.TF Colocalizes with PAR-2in IPF FibroblastsAs our results demonstrated a requirement for TF for FVIIa-induced PAR-2–dependent stimulation of HLF proliferation and ECM production,we sought to analyze TF expression in fibroblasts isolated from donor and IPF lungs.A pronounced increase in TF mRNA(Figure9A)and protein(Figures9B and 9C)expression was observed infibroblasts isolated from IPF lungs.These results were confirmed by immunofluorescence analysis(Figure9D).Moreover,laser-assisted microdissection in combination with qRT-PCR showed up-regulation of TF expression infibroblast foci of IPF lungs in comparison with alveolar septae of donor lungs(Figure E4).Immunohistochem-ical studies revealed colocalization of TF and PAR-2in fibroblasts/myofibroblasts in IPF lung tissue(Figure9E).Dual immunofluorescence staining of isolated IPFfibroblasts con-firmed colocalization of TF and PAR-2on the cell membrane (Figure9F).DISCUSSIONThere are several potential mechanisms by which activation of coagulation proteinases may contribute tofibrotic processes in acutely and chronically injured lungs.Excessive intraalveolar deposition offibrin is thought to inhibit surfactant function and to provide a provisional matrix on whichfibroblasts can pro-liferate and produce collagen(24).Furthermore,fibrin may serve as a reservoir of profibrotic growth factors(25).However, because lung injury and pulmonaryfibrosis occur infibrinogen-Figure4.Factor VIIa(FVIIa)–induced proliferation of human lungfibroblasts(HLFs)requires protease-activated receptor(PAR)-2and tissue factor (TF).(A)Determination of knockdown efficiency in HLFs after transfection with small interfering RNA(siRNA)against PAR-2(siPAR-2;top)or TF(siTF; bottom)as assessed by reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting.siR5scrambled siRNA.Data are representative of three independent experiments.(B)Effect of PAR-2and TF knockdown on donor HLF proliferation.Forty-eight hours after transfection with siR,siRNA against PAR-2(siPAR-2),and/or siRNA against TF(siTF),HLFs were stimulated for24hours in serum-free medium with FXa(50nM), FVIIa(50nM),or thrombin(65U/L)and cell proliferation was assessed by[3H]thymidine incorporation.The results are expressed as relative proliferation compared with unstimulated siR-transfected cells.Data represent mean values6SD from four independent experiments,each performed in triplicate.*P,0.05;**P,0.01;***P,0.001;NS5not significant.(C)Overexpression of PAR-2(left)and TF(right)in HLFs as assessed by Western blotting.Data are representative of four independent experiments.(D)Effect of PAR-2and TF overexpression on donor HLF proliferation.Forty-eight hours after transfection with empty vector(pcDNA3.1),PAR-2–expressing vector(pPAR-2)and/or TF-expressing vector (pTF)HLFs were stimulated for24hours in serum-free medium with FXa(50nM),FVIIa(50nM),or thrombin(65U/L)and cell proliferation was assessed by[3H]thymidine incorporation.The results are expressed as relative proliferation compared with unstimulated pcDNA3.1-transfected cells.Data represent mean values6SD from four independent experiments,each performed in triplicate.*P,0.05;**P,0.01;***P,0.001; NS5not significant.(E)Donor HLFs were treated for24hours in serum-free medium with FVIIa(50nM),inactivated FVIIa(FVIIi;50nM),peptide agonist for PAR-2(AP;100m M),or scrambled peptide control(RP;100m M).Cell proliferation was assessed by[3H]thymidine incorporation.The results are expressed as relative proliferation compared with unstimulated cells.Data represent mean values6SD from three independent experiments,each performed in triplicate.**P,0.01;***P,0.001;NS5not significant.(F)Donor and idiopathic pulmonaryfibrosis(IPF)fibroblasts were seeded in48-well plates,growth arrested in serum-free Dulbecco’s modified Eagle’s medium,and then stimulated for24hours in serum-free medium with FVIIa(50nM).Cell proliferation was assessed by[3H]thymidine incorporation.The results are expressed as relative proliferation compared with unstimulated donor cells.Data represent mean values6SD from four independent experiments,each performed in triplicate.*P,0.05;**P,0.01;NS5not significant.1708AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL1832011。
肿瘤基因表达调控
eIF4E
5’-cap
Coding region (‘open reading frame’)
STOP
3’-UTR RNARegulatory binding ‘element’: proteins
Stemloop binding proteins
13
Features of an mRNA
5’-UTR (untranslated region, ‘leader’) 5’-cap
Some mRNAs contain a stem loop(s) (inhibitory if stable) START
Coding region (‘open reading frame’)
Advantages: Speed of response – very quick Spatial control – e.g., in neurones, developing embryos, migrating cells
The fact that defects (mutations) in genes for proteins involved in mRNA transport and translation causes human diseases illustrates the crucial importance of correct mRNA localisation: e.g., FMRP/ fragile X protein (autism spectrum disorder) SMN (survival motor neuron; mutations/loss causes spinal muscular atrophy – affects motor neurons and causes infantile death)
肾衰宁胶囊治疗慢性肾衰竭钙磷代谢紊乱临床研究
肾衰宁胶囊治疗慢性肾衰竭钙磷代谢紊乱临床研究李任;徐永;陈轼【期刊名称】《浙江中西医结合杂志》【年(卷),期】2019(029)003【总页数】4页(P200-203)【关键词】慢性肾衰竭;钙磷代谢紊乱;肾衰宁胶囊;营养状况;肾功能【作者】李任;徐永;陈轼【作者单位】浙江省宁波市鄞州区鄞州第二医院药剂科, 宁波 315040;浙江省宁波市鄞州区鄞州第二医院药剂科, 宁波 315040;浙江省宁波市鄞州区鄞州第二医院药剂科, 宁波 315040【正文语种】中文慢性肾衰竭（chronic renal failure，CRF），临床特征以酸碱平衡和水电解质失衡，多种毒物、代谢产物蓄积，内分泌紊乱为主，是各种肾脏疾病末期的共同表现[1]。
作为慢性肾脏病（CKD）的常见并发症，慢性肾脏病矿物质和骨异常能够显著增加CKD患者心血管疾病的发生率，其中钙磷代谢紊乱是始动和加重条件[2]。
研究表明，CKD患者如长期伴有钙磷代谢紊乱，易引起全身血管钙化，导致病情不断发展、恶化，因此及早纠正钙磷代谢紊乱至为关键[3]。
目前，对钙磷代谢紊乱进行干预的常规药物主要包括降磷药物、拟钙剂及维生素D衍生物等，但部分西药价格昂贵，长期使用会产生不明确的副作用，因此在西药基础上结合中医是治疗CRF钙磷代谢紊乱更为有效的途径[4]。
本研究旨在观察肾衰宁胶囊联合碳酸钙治疗CRF血液透析伴钙磷代谢紊乱临床疗效及其对患者营养状况和肾功能的影响。
1 资料与方法1.1 临床资料选择2014年10月—2016年8月宁波市鄞州区鄞州第二医院收治的CRF血液透析伴钙磷代谢紊乱患者132例为研究对象，应用随机数字表法将其分为两组，对照组和观察组各66例。
本研究经医院医学伦理委员会审核通过，且所有患者均签署知情同意书。
1.2 纳入、排除标准纳入标准：（1）符合改善全球肾脏病预后组织（kidney disease：improving global outcomes，KDIGO）指南（2012年）[5]中慢性肾脏病、慢性肾脏病-矿物质和骨异常诊断标准及中华医学会肾脏病学分会制订的《血液净化标准操作规程》（2010版）[6]中终末期肾病透析指征；（2）出现肾脏结构或功能异常超过3个月患者，伴或不伴肾小球滤过率（glomerular filtration rate，GFR）降低，临床表现为病理异常或肾损害证据（血或尿成分异常或影像学检查异常）；（3）估算肾小球滤过率（eGFR）＜60mL·min-1·1.73m-2超过3个月患者，有或无肾损害指标；（4）钙（Ca）、磷（P）、甲状旁腺激素（iPTH）或维生素 D代谢异常患者，有或无骨矿化、转化，有或无骨骼线性生长、骨容量、骨强度异常，有或无血管或其他软组织钙化；（5）eGFR＜10mL·min-1·1.73m-2（非糖尿病肾病）或eGFR＜15mL·min-1·1.73m-2（糖尿病肾病）患者；（6）伴有发生感染、电解质紊乱、高血压、酸中毒且已得到有效控制的患者；（7）年龄18～65岁。
观察缬沙坦胶囊联合前列地尔注射剂和羟苯磺酸钙片治疗慢性肾衰竭（CRF）的临床疗效及安全性
观察缬沙坦胶囊联合前列地尔注射剂和羟苯磺酸钙片治疗慢性肾衰竭（CRF）的临床疗效及安全性发布时间：2021-04-09T07:40:32.401Z 来源：《健康世界》2021年1期作者：王庆梅[导读] 目的分析观察缬沙坦胶囊联合前列地尔注射剂和羟苯磺酸钙片治疗慢性肾衰竭（CRF）的临床疗效及安全性。
方法选取本院2018年09月-2019年10月期间收治的86例慢性肾衰竭患者进行此次研究，将其临床资料进行回顾性分析，并按照数字表法将所有患者均分为参照组和观察组两组，各43例。
王庆梅大庆油田五官医院 163000摘要：目的分析观察缬沙坦胶囊联合前列地尔注射剂和羟苯磺酸钙片治疗慢性肾衰竭（CRF）的临床疗效及安全性。
方法选取本院2018年09月-2019年10月期间收治的86例慢性肾衰竭患者进行此次研究，将其临床资料进行回顾性分析，并按照数字表法将所有患者均分为参照组和观察组两组，各43例。
其中使用前列地尔和羟苯磺酸钙片进行联合治疗的为参照组，在从参照组基础上使用颉沙坦胶囊进行治疗的为观察组，分析对比两组患者的临床治疗效果以及并发症状况。
结果观察组患者的并发症发生率低于参照组并且组间对比差异显著，具有统计学意义（P<0.05）；并且和参照组相比，观察组患者的治疗有效率相对较高，组间差异具有统计学意义（P<0.05）。
结论在对慢性肾衰竭患者所进行的治疗中，缬沙坦胶囊联合前列地尔注射剂和羟苯磺酸钙片的有效应用，可以进一步降低患者的并发症发生率，提高治疗效果，有着较高的安全性，有着推广价值。
关键词：缬沙坦胶囊；前列地尔注射剂；羟苯磺酸钙片；慢性肾衰竭[Abstract] Objective To analyze the clinical efficacy and safety of valsartan capsule combined with alprostadil injection and calcium dobesilate tablets in the treatment of chronic renal failure（CRF）.Methods 86 cases of chronic renal failure patients in our hospital from September 2018 to October 2019 were selected for this study.The clinical data were retrospectively analyzed，and all patients were divided into the reference group and the observation group according to the digital table method，with 43 cases in each group.The control group was treated with alprostadil and calcium dobesilate tablets，and the observation group was treated with jiesartan Capsule on the basis of the reference group.The clinical therapeutic effects and complications of the two groups were analyzed and compared.Results the incidence of complications in the observation group was lower than that in the reference group，and the difference between the two groups was statistically significant（P < 0.05）；and compared with the reference group，the effective rate of the observation group was relatively higher，and the difference between the two groups was statistically significant（P <0.05）.Conclusion in the treatment of patients with chronic renal failure，the effective application of valsartan capsule combined with alprostadil injection and calcium dobesilate tablets can further reduce the incidence of complications of patients，improve the treatment effect，with high safety，and has the promotion value.[Key words] valsartan capsule；alprostadil injection；calcium dobesilate tablets；chronic renal failure慢性肾衰竭是由于多种因素所造成的身体功能障碍以及身体功能异常的一种临床综合症，是各种慢性肾疾病共同作用所产生的结果【1】。
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Original articleExpression of osteopontin in chronic rhinosinusitis with and without nasal polypsChronic rhinosinusitis(CRS)is one of the most fre-quently reported chronic diseases,which is covering a spectrum of diseases,including nasal polyps(NPs)(1). The complexity of CRS makes the CRS etiology study and CRS therapeutics very difficult.Several hypotheses concerning its pathogenesis have been proposed,such as chronic microorganism infection,inhalant or food allergy,sinus ostial blockage,and local immunological disturbance(2).Although these studies have increased our understanding of CRS,the exact etiology of CRS is still far from completely revealed.So far,one of the most important characteristics of CRS is the prolonged and exaggerated inflammatory reaction in paranasal mucous membrane.As a result of the effect on inflammatory cell recruitment and activation,and tissue remodeling;cyto-kine and chemokine are implicated in the development of CRS(2).A lot of studies have reported increased levels of interleukin(IL)-4,IL-5,IL-13,IL-1b,and tumor necrosis factor(TNF)-a in the sinonasal mucosa from CRS patients with and without NPs(3,4).Recently,a novel cytokine,osteopontin(OPN),is catching scientistsÕeyes for its functions in recruiting inflammatory cells into the local inflammatory site and regulating the function of these cells,such as monocyte/ macrophage,dendritic cell,and T-cell(5–7).Osteopontin, also known as early T lymphocyte-activating gene-1and secreted phosphoprotein I,is a phosphorylated acidic arginine-glycine-aspartate(RGD)containing glycopro-tein that can bind certain CD44variants and integrin receptors and mediate cell-matrix interactions and cellu-lar signaling,involving both RGD-dependent and RGD-independent interactions(6).Expression of OPN is constitutive in bone and at epithelial surface and is upregulated in activated T-cells,macrophages,and tumor cells in model of inflammation,bone resorption,and tumor progression(5).Increasing evidence suggests that OPN may be tightly associated with inflammatory and immunological diseases.Osteopontin-deficient mice ex-hibit reduced immunity to viruses and other microorgan-isms(8).High levels of OPN have been observed in rheumatoid arthritis,CrohnÕs disease,and multiple scle-rosis(9–11).However,at present,the role of OPN in the human airway under normal and pathologic conditions has received little attention.Recently,two studies haveBackground:Osteopontin(OPN)is a multifunctional34-kDa extracellular matrix protein that can influence the inflammatory process.However,the presence of OPN in human sinonasal mucosa and its roles in the inflammatory process of chronic rhinosinusitis(CRS)are not clear.This study investigated the expression of OPN in human sinonasal mucosa,its cytokine-driven expression regulation,and its effect on cytokine production in sinonasal mucosa. Methods:Surgical samples were investigated by means of quantitative reverse transcriptase polymerase chain reaction for evaluation of OPN messenger RNA (mRNA)expression,and the presence and location of OPN protein expression were analyzed using immunohistochemistry.Furthermore,nasal explant culture was used to investigate the mutual regulatory interactions between interferon (IFN)-c,interleukin(IL)-4,IL-5,IL-13,IL-1b,and tumor necrosis factor (TNF)-a and OPN in sinonasal mucosa.Results:Osteopontin expression was significantly upregulated in CRS tissues com-pared with control tissues.There was a further significant increase of OPN expres-sion in patients with nasal polyps(NPs)and asthma.Immunohistochemistry revealed positive staining of OPN in epithelial cells,submucosal glands,infiltrating cells,and extracellular matrix.Osteopontin mRNA was induced by IFN-c,IL-1b, and TNF-a,but inhibited by IL-4and IL-13.On the contrary,OPN induced IFN-c,IL-4,IL-5,IL-13,IL-1b,and TNF-a production in sinonasal mucosa. Conclusions:The expression of OPN is upregulated in CRS.The mutual regu-latory interactions between OPN and inflammatory cytokines suggest that OPN may play an important role in the pathogenesis of CRS.X.Lu*,X.H.Zhang*,H.Wang,X.B.Long,X.J.You,Q.X.Gao,Y.H.Cui,Z.LiuDepartment of Otolaryngology-Head and Neck Surgery,Tongji Hospital,Tongji Medical College, Huazhong University of Science and Technology, Wuhan,P.R.ChinaKey words:chronic rhinosinusitis;nasal polyps; osteopontin;proinflammatory cytokine;regulation;Th1cytokine;Th2cytokine.Zheng LiuDepartment of Otolaryngology-Head and Neck SurgeryTongji Hospital,Tongji Medical College Huazhong University of Science and Technology 1095Jiefang AvenueWuhan430030P.R.China*Drs Lu and Zhang contributed equally to this article.Accepted for publication17May2008Allergy2009:64:104–111Ó2008The AuthorsJournal compilationÓ2008Blackwell MunksgaardDOI:10.1111/j.1398-9995.2008.01829.x104shown that OPN might be involved in the allergic lower airway disease (12,13).In the present study,we studied the possible role of OPN in CRS.Firstly,we examined the expression of OPN in sinonasal tissues from control and CRS patients by means of quantitative reverse transcriptase polymerase chain reaction (RT-PCR)and immunohistochemistry.Furthermore,we used nasal explant culture and quantitative RT-PCR technique to assess the potential regulatory axis between OPN and CRS-relevant inflammatory cytokines in sinonasal mucosa.Methods SubjectsThis study was approved by the ethical committee of Tongji Medical College of Huazhong University of Science and Technol-ogy and conducted with written informed consent from patients.Seventy-two patients who underwent functional endoscopic sinus surgery or septal surgery were enrolled for quantitative RT-PCR and immunohistochemistry experiments.These patients were divided into three groups:controls,CRS patients without NPs,and CRS patients with NPs.Patients undergoing septoplasty because of anatomic variations and not having any sinus disease were consid-ered control subjects and inferior turbinate mucosal samples were taken during surgery.Chronic rhinosinusitis with and without NPs was diagnosed according to the clinical criteria by Meltzer et al.on the basis of history,clinical examination,nasal endoscopy,and sinus-computed tomography scanning (14).Diseased sinus mucosal tissues and NP tissues were collected during surgery.Additional inferior turbinate mucosal samples of 22patients who underwent septal surgery and not having any sinus disease were obtained for nasal explant culture experiments.Clinical data of the patients are summarized in Table 1.In this study,subjects were excluded if they had received any oral steroid or antihistamine 3months before the surgery.Topical medications were withheld for a minimum of 1month before study.None had received antileukotrienes and immunotherapy.Patients who underwent previous sinus surgery were excluded.Medical management strategy was identical in both CRS groups.Human laryngeal squamous cell carcinoma tissues served as po-sitive controls for quantitative RT-PCR and immunohistochemistry experiments.Tissue preparation,immunohistochemistry,and quantificationSamples obtained during surgery were fixed in formalin and embedded in paraffin.Paraffin sections (4l m)were prepared from each block and stained with Giemsa.Protein expression of OPN was examined by means of immunohistochemical staining using the streptavidin-peroxidase complex method under the manufacturer Õs instructions (15).Sections were stained with anti-OPN monoclonal antibody (1:100,Santa Cruz Biotechnology,Santa Cruz,CA,USA).Color development was achieved with 3¢,3¢-diaminobenzidine which rendered positive cells brown.Species-and subtype-matched antibodies were used as a negative control.To examine the rela-tionship between OPN and eosinophil,the tissue sections positive for OPN staining were subjected to May-Gru nwald Giemsa (MGG)staining after quantification and digital photographs were taken.The same visual field pre-and post-MGG staining was compared.The number of total infiltrating cells,mononuclear cells,and eosinophils were determined by counting 10randomly selected fields in a blinded fashion at 400·magnification.Quantitative measure-ment of OPN protein expression was analyzed using the HPIAS-1000automated image analysis system as described elsewhere (Olympus,Tokyo,Japan;16).Ten microscopic fields were ran-domly selected from each slide under 400·magnification.Results were presented as 1/gray scores.The gray score reflects the optical density of selected field for analyzing (16).So,1/gray scores posi-tively correlated with the intensity of immunoreactivity.The gray score of background was measured in nontissue area and subtracted from the gray score of each selected field.Nasal explant cultureNormal inferior turbinate mucosal tissues were obtained during surgery and sectioned into multiple samples of approximately 6mm 3.One was processed for histologic evaluation and the others were used for tissue culture as described previously (16,17).Briefly,sections of tissue were placed on 0.4l m well inserts (Millipore Corp.,Billerica,MA,USA)in 2ml of tissue culture medium (18).The tissue was oriented with the epithelium being exposed to the air,forming an air–liquid interface to mimic the in vivo situation.Tissue was incubated in the absence or presence of various concentrations of interferon (IFN)-c (0.1,1,and 10ng/ml),IL-4(1,10,and 100ng/ml),IL-5(1,10,and 100ng/ml),IL-13(1,10,and 100ng/ml),IL-1b (1,10,and 100ng/ml),TNF-a (1,10,and 100ng/ml),or recombinant human osteopontin (rhOPN)(10nM,100nM,and 1l M)for various time durations between 4and 24h.All these cytokines were purchased from R&D Systems (Minneapolis,MN,USA).In some experiments,5l g/ml of actinomycin D (Sigma,St Louis,MO,USA)was added 12h after stimulation with IFN-c (10ng/ml)or IL-4(20ng/ml).Total RNAs were isolated immedi-ately (time =0)or at 4,8,12,and 24h after the addition of acti-nomycin D.The tissue was cultured at 37°C with 5%CO 2in humidified air.Quantitative reverse transcriptase polymerase chain reactionTissues were homogenized and RNA was extracted by using an RNeasy Mini kit (Qiagen,Valencia,CA,USA)and treated by using a DNA-free kit (Ambion,Austin,TX,USA)to remove contami-nating DNA.cDNA was reverse transcribed from 0.5l g of totalTable 1.Patients Õclinical dataQuantitative RT-PCR and immunohistochemistry Nasal explant culture Control groupCRS without NPs CRS with NPs No.of subjects 10323022Sex (male/female)6/419/1316/1414/8Age (years)18–4618–5916–6217–55No.of patients with asthma0580No.of patients with positive skin prick test results1213No.of patients with aspirin sensitivityCRS,chronic rhinosinusitis;NPs,nasal polyps;RT-PCR,reverse transcriptase poly-merase chain reaction.Osteopontin and chronic rhinosinusitisÓ2008The AuthorsJournal compilation Ó2008Blackwell Munksgaard Allergy 2009:64:104–111105RNA with random hexamer primers.Quantitative PCR was per-formed on the LightCycler system(Roche Diagnostics,Mannheim, Germany)by using the SYBR Premix Ex Taq kit(TaKaRa Bio-technology,Dalian,China)with the appropriate primers(Table2) and samples according to the manufacturerÕs protocol.In brief,1l l cDNA was added to10l l2·SYBR Premix Ex Taq master mix, 8l l RNase-free water,and1l l of each primer(5l M),resulting in a total volume of20l l.The PCR conditions consisted of an initial denaturation at95°C for30s,followed by amplification for45 cycles of5s at95°C and at different annealing temperature for20s. After PCR,a melting curve was constructed by increasing the temperature from65to95°C with a temperature transition rate of 0.1°C/s.Relative gene expression was calculated by using the comparative CT method(19).A sinus mucosa sample from one CRS patient without NPs was used as a calibrator in OPN mes-senger RNA(mRNA)expression study,whereas tissues without any treatment were employed as calibrators in the ex vivo culture study. Glyceraldehyde-3-phosphate dehydrogenase was used as a house-keeping gene for normalization and aÔno templateÕsample was used as a negative control.Statistical analysisData are presented as mean±SD.Paired sets of RT-PCR and immunohistochemistry data were compared with Kruskal–Wallis H and Mann–Whitey U-test.Paired t-test was used in tissue culture data analysis.Differences were considered statistically significant at a P-value of<0.05.ResultsOsteopontin expression in chronic rhinosinusitis patients with and without nasal polypsWe found that mRNA and protein expression of OPN was significantly upregulated in tissues from CRS patients compared with control tissues(P<0.01).Among CRS patients,patients with NPs showed significantly stron-ger mRNA and protein expression in sinonasal mucosa than patients without NPs of OPN(Fig.1A,B).When comparing asthmatic and nonasthmatic patients,we found a significant increase of OPN mRNA and protein expression in asthmatic patients in both CRS with and without NPs group.Moreover,asthmatic patients in CRS with NPs group had a significant higher expression of OPN than asthmatic patients in CRS without NPs group and a same trend was found when comparing nonasth-matic CRS patients with and without NPs(Fig.1C,D). No difference in OPN levels was found between atopic and nonatopic patients,and between male and female patients (data not shown).Osteopontin positive staining was demonstrated in epithelial cells,extracellular matrix, submucosal glands,and infiltrating cells by means of immunohistochemical staining(Fig.1E–J).Analyzing the relationship between OPN-staining intensity and the number of eosinophils,mononuclear cells,and total infiltrating cells in CRS patients with and without NPs, we only noticed a significant correlation between OPN expression and eosinophil cells in CRS patients with NPs (r=0.56,P<0.01).Employing OPN immunohisto-chemical staining and subsequent MGG staining,we found most eosinophils in lamina propria in NPs were OPN-positive cells(Fig.1K,L).The effect of cytokines on osteopontin messenger RNA production in sinonasal mucosaTo determine the potential factors contributing to the regulation of OPN gene expression in sinonasal mucosa, the modulation of OPN mRNA expression by selected Th1(IFN-c),Th2(IL-4,IL-5,and IL-13),and proin-flammatory cytokines(IL-1b and TNF-a)was examined in normal inferior turbinate mucosal tissue using ex vivo culture.Osteopontin mRNA expression was examined by means of quantitative RT-PCR.Interferon-c,IL-1b,and TNF-a were found to enhance the levels of OPN mRNA expression in a time-and dose-dependent manner (Fig.2A,B,G–J).The time course study showed the increase in OPN mRNA expression was detected4h after the addition of above cytokines,and the expression levels were almost close to the maximum around8–12h after stimulation.On the contrary,we found OPN mRNA expression levels were visibly inhibited by IL-4and IL-13 in a dose-dependent manner.The decrease in OPN expression was detected4h after stimulation with IL-4 and IL-13at10ng/ml and was nearly close to the maximum after12h stimulation in a time-dependent manner(Fig.2C–F).No obvious effect of IL-5on OPN mRNA expression was found(data not shown). Interferon-c and interleukin-4effect on osteopontin messenger RNA stabilityWe chose one Th1cytokine(IFN-c)as well as one Th2 cytokine(IL-4),which significantly influence the OPN mRNA expression to examine whether the regulation ofTable2.Primer sequences used for quantitative RT-PCR analysisPrimer SequenceAnnealing temperature(°C)OPN[s]5¢-CCAAGTAAGTCCAACGAAAG-3¢66[a]5¢-GGTGATGTCCTCGTCTGTA-3¢GAPDH[s]5¢-GAAGGTGAAGGTCGGAGTC-3¢66[a]5¢-GGAAGATGGTGATGGGATT-3¢IFN-c[s]5¢-GCTTTTCAGCTCTGCATCGTT-3¢66[a]5¢-TTTCTTAAGGTTTTCTGCTTCTTTTA-3¢IL-4[s]5¢-AACAGCCTCACAGAGCAGAAGAC-3¢66[a]5¢-GCCCTGCAGAAGGTTTCCTT-3¢IL-5[s]5¢-TAGCTCTTGGAGCTGCCTACGT-3¢60[a]5¢-CAAGGTCTCTTTCACCAATGCA-3¢IL-13[s]5¢-AGGATGCTGAGCGGATTCTG-3¢60[a]5¢-AAACTGGGCCACCTCGATT-3¢IL-1b[s]5¢-GATGAAAGACGGCACACC-3¢60[a]5¢-CTATGTCCCGACCATTGC-3¢TNF-a[s]5¢-TCTGGCCCAGGCAGTCA-3¢60[a]5¢-GCTTGAGGGTTTGCTACAACATG-3¢GAPDH,glyceraldehyde3-phosphate dehydrogenase;OPN,osteopontin;RT-PCR,reverse transcriptase polymerase chain reaction.Lu et al.Ó2008The Authors 106Journal compilationÓ2008Blackwell Munksgaard Allergy2009:64:104–111OPN gene expression in sinonasal mucosa by these cytokines occurs at the post-transcriptional level.Normal nasal tissues were stimulated with IFN-c(10ng/ml)or IL-4(20ng/ml)for12h,when the greatest change of OPN mRNA expression was noted.This was followed by the addition of5l g/ml actinomycin D.Total RNAs were isolated immediately(time=0)or at4,8,12,and24h after the addition of actinomycin D,and quantitative RT-PCR of OPN was performed.A decay curve was generated by plotting the ratios of normalized intensities relative to the respective samples at time=0.The results showed that in tissues treated with or without selected cytokine,no significant alteration was seen in decay kinetics of the OPN transcripts after the addition of actinomycin D(Fig.3).The effect of osteopontin on cytokine messenger RNA production in nasal mucosaTo explore the role of OPN in upper airway inflammatory network,the production of IFN-c,IL-4,IL-5,IL-13,IL-1b,and TNF-a induced by rhOPN was examined in normal inferior turbinate mucosa.We checked the concentration response and the time course of rhOPN on the mRNA expression of these selected cytokines, which was determined by means of quantitative RT-PCR. We found that all the cytokines mentioned above was induced by rhOPN in a time-and dose-dependent manner (Fig.4).Among these cytokines,IFN-c and IL-1b showed more notable up-regulation with approximate 10-fold increase.(Fig.4A,I).DiscussionAlthough OPN has been implicated in various immuno-logical events,its physiologic functions and potential roles in pathologic conditions remain to be defined. Currently,the evidence showing the relationship between OPN and human diseases is scarce.In the present study, we demonstrated for thefirst time that OPN expression was increased in CRS.Moreover,asthmatic patients hadA B CD EF GHFigure1.Expression of osteopontin(OPN)in sinonasal mucosa.(A–D)Quantification of OPN mRNA and protein expression in sinonasal mucosal tissues from controls and chronic rhinosinusitis(CRS)patients with and without nasal polyps(NPs)with quan-titative reverse transcriptase polymerase chain reaction analysis(A and C)and immunohistochemistry(B and D),respectively; #P<0.05and*P<0.01.(E–L)Representative photomicrographs of sinonasal tissue sections showing immunohistochemicalstaining.(E and F)OPN immunoreactivity in epithelial cells,extracellular matrix(E),and submucosal glands(F)of inferior turbinate mucosa from controls.(G and H)Obvious OPN immunoreactivity in epithelial cells,extracellular matrix(G),and submucosal glands (H)of sinonasal mucosa from CRS patients without NPs.(I and J)Obvious OPN immunoreactivity in epithelial cells,extracellular matrix(I),and submucosal glands(J)of NPs tissues.(K)OPN positive infiltrating cells in NPs tissues.(L)Same section as in(K)was restained with May-Gru nwald Giemsa(MGG).Most OPN positive cells in(K)could be identified as eosinophils by MGG staining. (original magnification:·400).Osteopontin and chronic rhinosinusitisÓ2008The AuthorsJournal compilationÓ2008Blackwell Munksgaard Allergy2009:64:104–111107Lu et al.A BC DE FG HI JFigure2.The effect of IFN-c,IL-4,IL-13,IL-1b,and TNF-a on osteopontin(OPN)mRNA expression in ex vivo cultured normal inferior turbinate mucosa.The tissues were incubated with selected cytokines at various concentrations for12h or at a certain concentration for various time durations.IFN-c(A and B),IL-1b(G and H),and TNF-a(I and J)enhanced,whereas IL-4(C and D) and IL-13(E and F)inhibited OPN mRNA expression in a time-and dose-dependent manner(n=6).#P<0.05and*P<0.01 compared with untreated mucosa.Ó2008The Authors 108Journal compilationÓ2008Blackwell Munksgaard Allergy2009:64:104–111higher levels of ONP expression.As a corollary,enhanced OPN expression was found in lung biopsies from asth-matics in two recent studies (12,13).We found that cells producing OPN in sinonasal mucosa included epithelial cells,submucosal glands cells,and infiltrating cells,which is in accordance with the finding in asthmatics (12,13).Interestingly,we found that CRS patients with NPs had a significantly stronger OPN expression in sinonasal tissuesthan CRS patients without NPs despite the coexistence of asthma or not,indicating OPN may have a higher impact on the pathogenesis of NPs.When we explored the correlations between OPN expression and different kinds of inflammatory cells in CRS with and without NPs,we could only find a significantly positive correlation between OPN expression intensity and the number of eosinophils in NPs.In NPs,there is apredominantA B C DE F G HI J K LFigure 4.Treatment with recombinant human osteopontin (rhOPN)induced a significant upregulation in expression of IFN-c (A and B),IL-4(C and D),IL-5(E and F),IL-13(G and H),IL-1b (I and J),and TNF-a (K and L)mRNA in ex vivo cultured normal inferior turbinate mucosa in a time-and dose-dependent manner (n =6).The tissues were incubated with rhOPN at various concentrations for 12h or at 100nM for various time durations.#P <0.05and *P <0.01compared with untreated mucosa.Osteopontin and chronic rhinosinusitisÓ2008The AuthorsJournal compilation Ó2008Blackwell Munksgaard Allergy 2009:64:104–111109eosinophilic infiltration.To explore whether eosinophils are OPN-expressing cells,we conducted immunohisto-chemical staining and subsequent MGG staining and found that most eosinophils were OPN expressing cells in NPs,suggesting the eosinophil is an important source of OPN production in NPs.Previous studies have demonstrated that the expres-sion of OPN in various mice models and cell lines could be modulated by several cytokines such as Th1(IFN-c), Th2(IL-4and IL-13),and proinflammatory cytokines (TNF-a and IL-1b)(5,20,21),but whether these cytokines are also involved in the OPN gene expression regulation in sinonasal mucosa is unclear.As these cytokines have been implicated in the pathogenesis of CRS(3,4),we evaluated the regulation of OPN gene expression in sinonasal mucosa by these cytokines.Our immunohistochemical study revealed that OPN was not only expressed by epithelial cells,but also by submu-cosal glands and inflammatory cells in sinonasal mucosa;therefore,we generated sinonasal mucosa explants and did ex vivo culture,which simulated the in vivo nasal environment.We found the mRNA expression of OPN was significantly induced by IL-1b, TNF-a,and IFN-c,but inhibited by IL-4and IL-13in a dose-and time-dependent manner.In line with our results,OPN expression was shown to be inhibited by IL-4and IL-13in monocytes and dendritic cells and be enhanced by IFN-c,IL-1b,and TNF-a in macrophages (20,21).As IL-1b and TNF-a have been reported to be are overexpressed in CRS(4),our results indicate that enhanced IL-1b and TNF-a may contribute to the up-regulated expression of OPN in CRS.Recently,Van Zele and Bruaene compared the difference in inflamma-tory mediators expression between different chronic sinus diseases and discovered that CRS without NPs revealed a Th1polarization with high levels of IFN-c, whereas NPs presented a Th2skewed eosinophilic inflammation with high IL-5,IL-13,and IgE concen-trations(3,22).Based on our presentfinding that OPN mRNA expression in sinonasal mucosa is induced by Th1cytokines,but inhibited by Th2cytokines,it is hard to explain why NPs with higher levels of Th2cytokines show stronger OPN expression compared with CRS without NPs with higher levels of Th1cytokines. However,it should be noted that although we discov-ered effect of these cytokines on OPN expression in vitro, their role in OPN gene regulation during the develop-ment of CRS in vivo still needs to be defined.On the contrary,besides the cytokines tested here,a lot of other inflammatory mediators involved in the patho-genesis of CRS may also contribute to the OPN gene regulation.Moreover,during the development of CRS in vivo,a lot of inflammatory cells will be recruited into sinonasal mucosa and may take part in OPN expression (22).Therefore,additional studies are required to investigate the mechanisms underlying the overexpressed OPN in CRS.It is well established that mRNA stability plays a central role in the regulation of gene expression.To explore the mechanisms underlying the effect of cytokines on OPN expression,we examined the mRNA stability of OPN after stimulated with IFN-c and IL-4.No significant alteration was seen in decay kinetics of the OPN transcripts after the addition of actinomycin D,suggesting that the regulation of OPN expression by IFN-c and IL-4is not mediated through the change of OPN mRNA stability.However, Konno et al.reported that faster decay of the OPN mRNA was seen in IL-4treated monocytes(20).This discrepancy could result from the difference between monocytes and inferior turbinate tissue.Further studies on signal trans-duction and transcript factors of OPN gene will be helpful to elucidate the mechanisms underlying cytokine-driven expression of OPN.Previous studies demonstrated that OPN is essential for IFN-a production by plasmacytoid dendritic cells(23). Osteopontin can induce IL-1b and TNF-a production in human monocytes,and IL-4,IL-13,IL-12,and IL-10 levels were decreased in draining lymph nodes of allergic mice treated with OPN-specific antibody(12,21).In the present study,we found that the mRNA expression of Th1,Th2,and proinflammatory cytokines could be induced by rhOPN in a time-and dose-dependent manner in sinonasal mucosa.It indicates that OPN may play a pivotal proinflammatory function in the inflammatory and immunological process in sinonasal mucosa.Previ-ous studies have shown that OPN is a multifunctional molecule favoring Th1immune response and has been considered as a Th1cytokine by some scientists(5).Our present and recent studies from others indicate that OPN may have a wide range of functions and also be deeply involved in the Th2-derived diseases(12,13).Very interestingly,although we found OPN could induce Th2cytokines mRNA expression in nasal mucosa, Th2cytokines were shown to inhibit OPN mRNA expression,indicting a possible negative feedback loop may exit between OPN and Th2cytokines in nasal airway inflammatory process.Normal inferior turbinate tissue consists of epithelium, submucosal glands,infiltrating cells,fibroblasts,and extracellular matrix.Although ex vivo culture experiment can mimic the in vivo nasal environment,it is hard to tell which component exerts major effect using this study method.Further study employing specific cell lines is needed to deepen our understanding of OPNÕs function in CRS.ConclusionsThe expression of OPN is upregulated in CRS with and without NPs.There was a significant further increase of OPN expression in patients with NPs and asthma.The mutual regulatory interactions between OPN and inflam-matory cytokines in sinonasal mucosa suggest that OPNLu et al.Ó2008The Authors 110Journal compilationÓ2008Blackwell Munksgaard Allergy2009:64:104–111may play an important role in the pathogenesis of upper airway diseases.However,further studies are warranted to reveal the exact role of OPN in CRS with and without NPs. AcknowledgmentsThis study was supported by National Nature Science Foundation of China(NSFC)grant30500557,scientific research foundation for the returned overseas Chinese scholars of State Education Ministry (SRF for ROCS,SEM)[2006]331,and program for New Century Excellent Talents in University from State Education Ministry (NCET-07-0326to Dr Zheng Liu).Conflict of InterestNoneReferences1.Kaliner MA,Osguthorpe JD,FiremanP,Anon J,Georgitis J,Davis ML et al.Sinusitis:bench to bedside–currentfindings,future directions.J Allergy Clin Immunol1997;99:S829–S848.2.Fokkens W,Lund V,Mullol J.EuropeanPosition Paper on Rhinosinusitis andNasal polyps group.European position paper on rhinosinusitis and nasal polyps 2007.Rhinol Suppl2007;20:1–136.3.Van Zele T,Claeys S,Gevaert P,VanMaele G,Holtappels G,VanCauwenberge P et al.Differentiation of chronic sinus diseases by measurement of inflammatory mediators.Allergy2006;61:1280–1289.4.Lennard CM,Mann EA,Sun LL,Chang AS,Bolger WE.Interleukin-1b, interleukin-5,interleukin-6,interleukin-8,and tumor necrosis factor-a inchronic sinusitis:response to systemiccorticosteroids.Am J Rhinol2000;14:367–373.5.Denhardt DT,Noda M,OÕRegan AW,Pavlin D,Berman JS.OPN as a means to cope with environmental insults:reg-ulation of inflammation,tissue remod-eling,and cell survival.J Clin Invest2001;107:1055–1061.6.Zhu B,Suzuki K,Goldberg HA,Rittling SR,Denhardt DT,McCulloch CA et al.OPN modulates CD44-dependent chemotaxis of peritonealmacrophages through G-protein-cou-pled receptors:evidence of a role for an intracellular form of OPN.J Cell Phys-iol2004;198:155–167.7.OÕRegan AW,Nau GJ,Chupp GL,Berman JS.OPN(Eta-1)in cell-mediated immunity:teaching an old dog new tricks.Immunol Today2000;21:475–478.8.Patarca R,Saavedra RA,Cantor H.Molecular and cellular basis of genetic resistance to bacterial infection:the role of the early T-lymphocyte activation-1/ osteopontin gene.Crit Rev Immunol1993;13:225–246.9.Hur EM,Youssef S,Haws ME,ZhangSY,Sobel RA,Steinman L.Osteopon-tin-induced relapse and progression ofautoimmune brain disease through en-hanced survival of activated T cells.NatImmunol2007;8:74–83.10.Gassler N,Autschbach F,Gauer S,Bohn J,Sido B,Otto HF et al.Expres-sion of osteopontin(Eta-1)in Crohndisease of the terminal ileum.Scand JGastroenterol2002;37:1286–1295.11.Xu G,Nie H,Li N,Zheng W,Zhang D,Feng G et al.Role of osteopontin inamplification and perpetuation of rheu-matoid synovitis.J Clin Invest2005;115:1060–1067.12.Xanthou G,AlissafiT,Semitekolou M,Simoes DCM,Economidou E,Gaga Met al.Osteopontin has a crucial role inallergic airway disease through regula-tion of dendritic cell subsets.Nat Med2007;13:570–578.13.Kohan M,Bader R,Puxeddu I,Levi-Schaffer F,Breuer R,Berkman N.Enhanced osteopontin expression in amurine model of allergen-induced air-way remodeling.Clin Exp Allergy2007;37:1444–1454.14.Meltzer EO,Hamilos DL,Hadley JA,Lanza DC,Marple BF,Nicklas RAet al.Rhinosinusitis:establishing defini-tions for clinical research and patient-care.J Allergy Clin Immunol2004;114:155–212.15.Liu Z,Lu X,Wang H,Gao Q,Cui Y.The up-regulated expression of tenascinC in human nasal polyp tissues is relatedto eosinophil-derived transforminggrowth factor beta1.Am J Rhinol2006;20:629–633.16.Liu Z,Lu X,Wang H,Gao Q,Cui Y.Group II subfamily secretory phospho-lipase A2enzymes:expression in chronicrhinosinusitis with and without nasalpolyps.Allergy2007;62:999–1006.17.Hauber HP,Daigneault P,Frenkiel S,Lavigne F,Hung HL,Levitt RC et al.Niflumic acid and MSI-2216reduceTNF-alpha-induced mucin expression inhuman airway mucosa.J Allergy ClinImmunol2005;115:266–271.18.Sha Q,Truong-Tran AQ,Plitt JR,BeckLA,Schleimer RP.Activation of airwayepithelial cells by toll-like receptor ag-onists.Am J Respir Cell Mol Biol2004;31:358–364.19.Livak KJ,Schmittgen TD.Analysis ofrelative gene expression data using real-time quantitative PCR and the2(-Del-taDelta C(T))method.Methods2001;25:402–408.20.Konno S,Eckman JA,Plunkett B,Li X,Berman JS,Schroeder J et al.Interleu-kin-10and Th2cytokines differentiallyregulate OPN expression in humanmonocytes and dendritic cells.J Inter-feron Cytokine Res2006;26:562–567.21.OÕRegan A,Berman JS.Osteopontin:akey cytokine in cell-mediated andgranulomatous inflammation.Int JExp Pathol2000;81:373–390.22.Van Bruaene N,Perez-Novo CA,Basinski TM,Van Zele T,Holtappels G,Ruyck ND et al.T-cell regulationin chronic paranasal sinus disease.J Allergy Clin Immunol2008;121:1435–1441.23.Shinohara ML,Lu L,Bu J,WerneckMB,Kobayashi KS,Glimcher LH et al.Osteopontin expression is essential forthe interferon-alpha production byplasmacytoid dendritic cells.NatImmunol2006;7:498–506.Osteopontin and chronic rhinosinusitisÓ2008The AuthorsJournal compilationÓ2008Blackwell Munksgaard Allergy2009:64:104–111111。