Expression and significance of vascular endothelial growth factor D in gastric cancer

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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]。

缺氧对肝泡状棘球蚴原头节血管内皮生长因子和CD34表达的影响研究

1702 • http ： //www. chinagp. net E-mail ： zgqkyx@ chinagp. net, cn缺氧对肝泡状棘球蚴原头节血管内皮生长因子和〔〇34表达的影响研究张怀，张示杰*，陈骞，王岩，苏争明，吴向未，孙红论著【摘要】目的探讨体外缺氧条件下肝泡状棘球蚴原头节中血管内皮生长因子（VEGF )、CD 34表达水平的变化。

方法2016年4一 10月，选取实验用腹腔感染肝泡状棘球蚴18周的长爪沙鼠30只，采用颈椎脱臼法处死，选取光滑、成簇的游离于腹腔或附着在腹壁、肠系膜、肝表面的肝泡状棘球蚴组织。

培养肝泡状棘球蚴原头节，分为两组，对照组和实验组（给予含100|xm 〇l/L 二氯化钴的RPMI 1640培养基），培养第0、6、12、18、24h 倒置显微镜下观察肝泡状棘球蚴原头节活力。

培养1、24 h 采用酶联免疫吸附试验（ELISA )法检测肝泡状棘球蚴原头节中缺氧诱导因子la (H IF -la )、VEGF 、CDM 表达水平，Western blotting 法检测肝泡状棘球蚴原头节中VEGF 、CDM 的表达。

结果培养0、6、12、18、24 h ，对照组与实验组肝泡状棘球蚴原头节活力比较，差异均无统计学意义（P >0. 05)。

培养 12、24h ，实验组肝泡状棘球蚴原头节H IF-la 、VEGF 、CDM 表达水平较对照组升高（P <0.05)。

结论缺氧可以使肝泡状棘球蚴原头节中VEGF 和CDM 表达水平升高，这可能是肝泡状棘球蚴病呈浸润性生长的重要机制之一。

【关键词】棘球蚴病，肝；缺氧；血管内皮生长因子类；CD m 【中图分类号】R 532. 32 【文献标识码】 A D 0I ： 10. 3969/j. i n . 1007 -9572. 2017. 14. 011张怀，张示杰，陈骞，等.缺氧对肝泡状棘球蚴原头节血管内皮生长因子和CDM 表达的影响研究[J ].中国全科医学，2017，20 (14) : 1702 - 1706. [ www. chinagp. netZHANG H, ZHANG S J, CHEN Q, et al. Effect of anoxia on expression of vascular endothelial growth factor and CD 34 in hepatic alveolar echinococcosis protoscolex [J]. Chinese General Practice, 2017, 20 (14) : 1702 -1706.nese General .Mfect of Anoxia on Expression of Vascular Endotlielial Growth Factor and CD 34 in Hepatic Alveolar Echin Protoscolex ZHANG Huai , ZHANG S h i-jie \ CHEN Qian , WANG Yan , SU Zheng-m ing , W UXiang-wei , SUN Hong The First Affiliated Hospital , School of Medicine , Shihezi University , Shihezi 832000 , China '* Corresponding author ： ZHANG Shi - jie, , P rofessor , Main research directions ： hepatobiliary surgery ； E-mail ： zhangshijiewk1 @ sinn. com【Abstract 】 Objective To investigate the expression level of vascular endothelial growth factor (VEGF) and CD 34 in t e hepatic alveolar echinococcosis protoscolex under the condition of vitro anoxia. Methods From April to October in 2016 , the 30 cases gerbils of abdominal infection with hepatic alveolar echinococcosis for 18 weeks were selected for experiment , and were sacrificed by cervical dislocation. The smooth clusters of hepatic alveolar echinococcosis tissues isolated in abdom or tissues adhere to the abdominal wall , mesentery , liver surface were selected. The hepatic alveolar echinococcosis protoscolex was cultured to divide into control group and experiment group ( RPMI 1640 medium containing 100 ^mol/]L cobalt chloride). The hepatic alveolar e chinococcosis protoscolex activity was observed by inverted microscope at the beginning , and the 6th ,12th , 18th , 24th hour of the culture process. At the 12th and 24th hour of the culture process , enzyme - linked immunosorbent assay (ELISA) was used to detect the expression level of hypoxia - inducible factor 1 alpha (H IF -la) , VEGF and CD 34 in hepatic alveolar echinococcosis protoscolex. Western blotting method was applied to detect the e xpression o f VEGF and Results The echinococcosis protoscolex activity at the 0th , 6th , 12th , 18th , 24th hour of the culture process was not significantly different between control group and experiment group ( P > 0. 05 ) . Compared with the control group , the expression level of HIF-la , VEGF , CD 34 were significantly increased at the 12th and 24th hour of the culture process in the experiment group (P <0. 05 ). Conclusion Hypoxia can increase the expression l evel of VEGF and CD 34 in echin基金项目：国家自然科学基金资助项目（81560518)832000新疆石河子市，石河子大学医学院第一附属医院*通信作者：张示杰，教授，主要研究方向：肝胆外科；E-mail ：zhangshijiewkl@and this may be one of the important mechanisms of invasive growth presented by hepatic alveolar echinococcosis.【K e yw o r d s 】 Echinococcosis ，hepatic ； Anoxia ； Vascular endothelial growth factors ； CD 34中国 pmaip 字2017年5月第20卷第14期http ； //www. chinagp. net E-mail ； zgqkyx@ chinagp. net, cn • 1703 •◊本研究背景及创新点：血管内皮生长因子（VEGF )是血管新生的关键递质之一，CD3为公认的标记肝癌新生血管特异性最好的指标之一；实验研究发现缺氧引起的血管新生是肝癌浸润性生长的重要原因；因此VEGF 、 CD %被广泛应用于肝癌的研究中。

MusclesofFacialExpression

MusclesofFacialExpressionMuscles of the mouthThe Orbicularis Oris Muscle are responsible for closing your mouth, kissing and pouting as well as blowing a horn Orbicularis Oris MuscleThe Orbicularis Oris is the muscle responsible for closing your mouth, kissing and pouting. As the name suggests the fibbers of the orbicular oris muscle encircle the mouth is a sphincter muscle. It has an origin points at the maxilla and the the mandible the lips and the buccinator muscles and it’s insertion points encircle the mouth.Buccinator MuscleThe buccinator is the muscle of the cheek and assists in chewing motion by holding the cheek close to the teeth. It is also the muscle used for blowing such as playing a trumpet. It has origin points at the maxilla, the mandible, and the superior constrictor pharyngis muscle. The insertion points are the orbicularis oris and the modiolus, beneath the risorius muscle.Levator Anguli Oris MuscleThe Levator Anguli muscle assist the naso-labial fold in the cheek. It lifts the upper lip to expose the teeth when smiling. The origin point is on the maxilla just below the infraorbital foramrn and has an insertion point at the modiolus.These muscles combined are responsible for laughing and smilingZygomaticus Major MusclesThe zygomaticus major muscle works with the risorious muscle to assist laughing and smiling by lifting the corners of the mouth. It’s origin point id from the Zygomatic bone and hasinsertion points at the orbicularis at the modiolus.Zygomaticus Minor MuscleThe zygomaticus minor muscle also works with the risorius muscle lifting the lip to assist laughing and smiling. It’s origin points from the malar surface of the zygomatic bone and has insertion points on the orbicularis oris just next to and above the zygomaticus major .Levator Labii Superior MuscleThe levator labii superioris muscle is one of the muscles responsible for lifting the upper lip. It has origin points on the cheek bone near where it meets the bones of the nose and insertion points into the skin of the nose and skin of the lip.Levator Anguli MuscleThe name of the levator anguli oris muscle lifts the mouth at corners. It has origin points just outside and beneath the orbital rim and insertion points at the modiolus. It lies beneath them, coursing under the levator labii superioris, then arising just outside of and beneath the orbital rim.Levator Labii Superior Alawque Nasi MuscleThe levator labii superioris alaeque nasi muscle is responsible for the facial expression allowing you to sneer. It has an origin point from the upper frontal process of the maxilla and insertion points at the skin of the lateral part of the nostril and upper lip Depressor Anguli Oris MuscleThe depressor anguli oris muscle is located on the lower lip and aids in drawing the lower lip downward. It has origin points out of the fibers of the platysma muscle and has insertion points at the modiolus, mingling its fibers with the risiorious and the orbicularis oris,Depressor Labii Inferior MuscleThe deppressor labii inferioris muscle is primarily responsible for depressing or drawing down muscle of the lower lip. It’s origin point is from the mental region of the lower mandible and has an insertion points on the orbicular oris.Risorious MuscleThe risorious muscle is primarily responsible for the facial expressions of laughing and smiling. It’s origin point is in the fascia of the cheek and has insertion points into the orbicular oris.。

肺鳞癌中Furin和E-cadherin的表达研究

²临床论著²肺鳞癌中Furin和E-cadherin的表达研究牛灵1陶琳2董雅璐3【摘要】目的检测肺鳞癌中弗林蛋白酶（Furin）和上皮型黏附素（E-cadherin）的表达，分析二者在不同临床特征中的表达差别，关注其临床意义。

方法选择65例肺鳞癌作为观察组，选择50例正常肺组织作为对照组，应用免疫组化方法检测Furin和E-cadherin的表达，统计学分析应用卡方检验或相关性分析。

结果观察组中Furin表达的阳性率明显高于对照组（53.85% vs. 4.00%，P＜0.000 1），观察组中E-cadherin表达的阳性率明显低于对照组（16.92% vs. 98.00%，P＜0.000 1）。

观察组中Furin和E-cadherin的表达均与肿瘤的最大径、分化程度和脉管浸润密切相关（P＜0.05）。

察组中Furin和E-cadherin的表达无相关性（P＞0.05）。

结论肺鳞癌中Furin高表达、E-cadherin低表达是促进肿瘤发生和进展的重要因素，Furin和E-cadherin无明显的协同作用。

【关键词】肺；费林蛋白酶；免疫组织化学；上皮型黏附素；鳞癌Study on expressions of Furin and E-cadherin in lung squamous cell carcinoma Niu Ling1, Tao Lin2,Dong Yalu3. 1Department of Respiratory, Xinjiang People’s Hospital, Urumqi 830002, China; 2The SecondMedical Department of Midong Branch, Xinjiang People’s Hospital, Urumqi 830000, China; 3Departmentof Oncology, Lanzhou Military Region Urumqi General Hospital, Urumqi 830000, ChinaCorresponding author: Niu Ling, Email: 363141843@【Abstract】Objective To detect the expressions of Furin and E-cadherin in lung squamous cellcarcinoma, analyze the clinical significance. Methods The observation group included 65 cases with lungsquamous cell carcinoma, the control group included 50 cases with normal lung tissue. Expressions ofFurin and E-cadherin were detected by IHC. Chi-square test and correlation analysis were used in study.Results The expression of Furin was higher in the observation group than in the control group (53.85%vs. 4.00%, P<0.000 1). The expression of E-cadherin was lower in the observation group than in the controlgroup (16.92% vs. 98.00%, P<0.000 1). Expressions of Furin and E-cadherin were associated with diameter,differentiation and vascular invasion (P<0.05). The expressions of Furin and E-cadherin were not correlated inobservation group (P>0.05). Conclusion The higher expression of Furin, lower of E-cadherin maypromote the occurrence and development of lung squamous cell carcinoma. The expressions of Furin andE-cadherin are not obvious synergistic effect in observation group.【Key words】Lung; Furin; Immunohistochemistry; E-cadherin; Squamous cell carcinoma肺癌中以鳞癌最常见，其发病时进展快，预后不理想[1]。

2007 Targeted expression of vascular endothelial growth factor 165 in

Brief reportTargeted expression of vascular endothelial growth factor 165 in the hrDNA locus mediated by hrDNA targeting vectorLIANG Zong-min, LUO Hong, YANG Yi-feng, LIU Xiong-hao, XIE Li, XUE Zhi-gang, HU Dong-xu, LIANG De-sheng, XIA Kun and XIA Jia-huiKeywords: human-derived vector; site-specific integration; targeting expression; vascular endothelial growth factor 165ascular endothelial growth factor (VEGF) is an angiogenic regulator that stimulates endothelial cell migration, proliferation, and angiogenesis. VEGF gene therapy is a new promising approach to induce therapeutic angiogenesis for the treatment of ischemic myocardial and limb diseases. Recently, clinical studies have demonstrated successful outcomes using plasmids, retroviruses and adenoviruses.1-3But the safety of those vectors is poor, which has become a serious concern.Besides immunogenicity caused by viral vectors, a furtherproblem is that viral vectors show a preference to integrate into the transcription or control region of active genes, which may induce tumors and other disorders.4-6Efficient and safe nonviral vectors have attracted moreand more attention. The researchers from the NationalLaboratory of Medical Genetics of China have constructed a new nonviral vector ,pHrneo. This is a human source gene vector that targets heterologous genesto the human ribosomal DNA (hrDNA) locus with anefficiency of integration up to 10-4-10-5. In this study,pHrneo was chosen as the vector in the research of genetherapy with VEGF165.METHODSMaterials Restriction enzymes were purchased from TaKaRa, Japan and Fermentas, USA. TcGb, pHrneo were constructed by National Laboratory of Medical Genetics of China. Reverse Transcription System was obtained from Promega, USA. HT-1080 cells were purchased from ATCC. Anti-human VEGF (mouse anti-human) was purchased from ABcam (ab1316, England) and anti-GFP was from Sigma (G1544, USA). HRP IgG (rabbit anti- mouse) was obtained from PIERCE Co (31457, USA). The ELISA kit was purchased from R&D (DVE00, USA).Construction of pHrneo-VEGF165 Using brain cell cDNA as templates, a pairs of primers (VEGF165, forward 5’-GCTAGCGCCGGGAGGAAGA- GTAGC-3’, reverse 5’-GCTAGCTCTGTCGATGGT- GATGGTGT-3’) were designed to generate an 800 bpPCR fragment. Then another pair of primers (VEGF165 sense: 5’-AACCCGGGATGAACTTTCTGCTGTCT- TG-3’, VEGF165-antisense: 5’-GGACCGGTCGCCTC- GGCTTGTCACATCTG-3’) were used to generate a 580 bp PCR fragment of VEGF165 using the first PCR products as templates. VEGF165 PCR products were reclaimed and cloned into a pGEM-T vector (T-VEGF165) then sequenced with a ABI377 DNA analyzer. The VEGF165 fragment was subsequently cloned into a Nhe I site of pHrneo to generate the targeting vector,pHrneo-VEGF165 (Fig. 1).Transfection of pHrneo-VEGF165 plasmid HT1080 cells were grown in DMEM containing 15%foetal calf serum (FCS) at 37˚C under 5% CO 2. HT1080 cells (2×106) were stably transfected by electroportion with pHrneo-VEGF165 (20 µg DNA) linearized by Ahd I. Parameters of electroportion: 2.0 kV , 50 µF. The transfected HT1080 cells were applied to four 10 cm-diameter dishes containing 10 ml of medium per plate.Twenty-four hours after transfection, the medium was removed and replaced by fresh medium. 24-hour later a fresh medium containing 0.3 mg/ml G418 was added to replace the medium. The medium containing 0.3 mg/ml G418 was refreshed every three days. After two weeks individual drug-resistant colonies were picked, transferred into 12-well plates and expanded into cell lines.PCR identification of the site-integration clones Genomic DNA was extracted from individual drug-resistant colonies by a miniprep procedure involving sodium dodecyl sulfate lysis, proteinase K digestion, phenol extraction and ethanol precipitation. PCR was performed to identify the site-integration clones using genomic DNA as templates. Primer Screen-EC (5’-GGGTGGGGCAGGACAGCAAGGGGGAGGA T-3’) Department of Cardiovascular Surgery, Second Xiangya Hospital of Central South University, Changsha 410011,China (Liang ZM,Yang YF, Xie L and Hu DX) National Laboratory of Medical Genetics of China, Changsha 410011,China (Liu XH, Xue ZG ，Liang DS, Xia K and Xia JH)Department of Respiratory Medicine, Second Xiangya Hospital of Central South University, Changsha 410011,China (Luo H) Correspondence to: Dr. YANG Yi-feng, Department of Cardiovascular Surgery, Second Xiangya Hospital of Central SouthUniversity, Changsha 410011, China (Tel: 86-731-5295801. Email: yyf627@)LIANG Zong-min and LUO Hong are equal contributors.This study was supported by a grant from the National Natural Science Foundation of China (No. 30270734).VFig.1. Construction of targeting vector, pHrneo-VEGF165. A580bp fragment of VEGF165 was amplified by PCR, thencloned into pGEM-T and verified by DNA sequencing. The VEGF165 fragment was cut from T-VEGF165 by Age I and Sma I , and was inserted into TcGb that had been opened byAge I and Sma I to construct TcGb-VEGF165; the VEGF165expression cassette was cut from TcGb-VEGF165 by Nhe Ithen was inserted into a Nhe I site of pHrneo as the targetingvector: pHrneo-VEGF165. TcGb vector contains CMV , EGFP and BGHpoly(A).pHrneo is the hrDNA targeting vector. LHA:long homologous arm;SHA: short homologous arm;EMCV-IRES: internal ribosome entry site element ofencephalomyocarditis virus. In pHrneo, NEO lacks apromoter and is expressed from the promoter of the hrDNAgene following homologous recombination. located in BGH poly(A) and primer screen-dn (5’-GGCGATTGATCGGCAAGCGACGCTCAGACAG- 3’) located out of the homologous arm of hrDNA locus were used to generate a 1.4 kb DNA product. The reaction program is 97˚C for 5 minutes; 97˚C for 30 seconds, 72˚C for 2.5 minutes for 10 cycles (-0.6˚C /cycle); 97˚C for 30 seconds, 66˚C for 30 seconds, 72˚C for 2 minutes for 25 cycles; 72˚C for 10 minutes. PCR fragments were analyzed on an ABI377 DNA sequencing instrument. RT-PCR analysis for mRNA of VEGF165 in the site-specific integration clones Total cellular RNA was isolated from the site-integration cloned cells with TRIzol reagent. One microgramme of each RNA sample was reverse transcribed using Reverse Transcription System after determination of the RNA concentrations. A total of 100 ng of the reverse transcript was used to amplify a fragment of about 1.3 kb by PCR with primers VEGF165-up (5’-AACCCGG- GATGAACTTTCTGCTGTCTTG-3’) and EGFP-down (5’-CAGGCTTGCTTTACTTGTACAG-3’). PCR conditions were 95˚C for 5 minutes ；94˚C for 30 seconds, 57˚C for 20 seconds, 72˚C for 40 seconds for 32 cycles then 72˚C for 5 minutes.Western blot analysisWestern blot analysis was performed to verify VEGF165 fusion protein expression in site-integration clonal cells. Individual site-specific integrated clonal cells were cultured in DMEM with 15% serum. At 90% confluence, cells were harvested and lysed with 5×loading solution. Samples were separated on 8% SDS-PAGE gels and electroblotted onto a PVDF membrane that was then probed with specific antibodies and visualized by the ECL system. For VEGF the mouse anti-human VEGF and mouse anti-GFP were used.ELISA assays for human VEGF165 antigenThe human VEGF165 antigen was quantified in the supernatants (with 1% serum) of individual site-specific integration clonal cells using an ELISA kit according to the manufacturer’s instructions. The microtiter plates were first coated with antibodies against VEGF and the concentrations of VEGF were obtained from the standard curve. RESULTSConstruction of pHrneo-VEGF165 A 580bp PCR fragment of VEGF165 was generated by PCR using primers VEGF165-up and VEGF165-down. The fragment was identified by DNA sequencing. pHrneo-VEGF165 can be excised into two fragments:8840 bp and 3948 bp. Identification of site-specific integration clones by PCR and efficiency of site-specific integration.When homologous recombination occurred, the fragment within the two homologous sequences of pHrneo- VEGF165 could be targeted into the homologous sequence of the HT1080 genome. Using genomic DNA prepared from individual drug-resistant colonies by a miniprep procedure as templates, a specific fragment of 1.4 kb could be amplified by PCR with the primer Screen-EC and primer Screen-dn. Positive results and the 1.4 kb fragment were shown to be the target fragment by DNA sequencing (Fig. 2). In our research, 2×106 cells were transfected and half (50%) of them died 24 hours after transfection. There were about 200 individual drug-resistant clones. We found 10 site-specific integration clones through analyzing 26 individual drug-resistant clones. The efficiency of site-specific integration was 7.7×10-5. RT-PCR analysis RT-PCR was performed to examine the mRNA for the fusion protein extracted from site-specific integration clones. The forward primer was VEGF165-up and the reverse primer was GFP-down, the 1.3 kb PCR productFig. 2. Identification of site-specific integration clones. A1.4-kb specific fragment was amplified from gDNA ofsite-specific integration clones. M: DNA marker; Lines 2-5, 7,8: site-specific integration clones; line 6: site-unspecific integration clones ; line 1: control. was the amplification of VEGF165-EGFP fusion protein. A specific fragment of 1.3kb was generated in all positive clones (Fig. 3A). Transfection of the pHrneo-VEGF165 plasmid Cell transfection was carried out using electroportion. Two weeks after transfection, individual drug-resistant colonies formed and the fusion protein, which consists of VEGF165 protein and green fluorescent protein (GFP), was expressed. The fusion protein was detected under a fluorescence microscope (Fig .3B). Western blotting The fusion proteins translated from mRNA of VEGF165 (576 bp) and EGFP (720 bp) were all detected in site-specific integration with anti-human VEGF and anti-GFP antibodies. In HT1080 cells a single band at approximately 40 kD (VEGF165 is a glycoprotein consisting of two identical polypeptide chains) was seen when using anti-human VEGF antibody, detecting the secretion of endogenous VEGF protein. No band was detected with anti-GFP antibody. In site-specific integration cloned cells, a major band at approximately 96 kD was detected using anti-GFP antibody (dimers of the fusion protein) and with the anti-human VEGF antibody two bands were detected of approximately 96kD (fusion protein) and approximately 40 kD(endogenous VEGF protein from HT1080). (Fig. 3C). Quantity of VEGF165 antigenFour site-specific integration clones were tested with the ELISA method. The fusion protein of VEGF165 rangedfrom 400 to 1400 pg ·10-6 cells ·24 h -1, the average expression level of the 4 experiments was (1000±23)pg ·10-6 cells ·24 h -1.DISCUSSIONpHrneo-VEGF165 was constructed successfully and targeted into the hrDNA locus of HT-1080 cells. The site-specific integration clones were picked out in this study. Human ribosomal RNA genes (rDNA) are located in the D and G group chromosome. In human cells there are about 600-800 copies of rDNA per cell.7 For an exogenous gene to be inserted into a ribosomal gene target site to replace a copy of rDNA there may be no effect on the cell’s physiological function. A heterogenous DNA site-specific targeting into an rDNA locus was first performed in saccharomyces 8 and also achieved successfully in trypanosoma cruzi.9 However, the recombination hotspots in rDNA regions have not been found in mammalian cells till to now. Many experiments on gene targeting indicate that the frequency of homologous recombination in mammalian cells is much lower than that in prokaryotes and other eukaryotes with an efficiency of about 10-6-10-8. Absolute frequency of homologous recombination in somatic cells was some two orders of magnitude lower than in ES cells.10 High efficiency (7.7×10-5) of homologous mitotic recombination was observed in this study and it suggested that the new vector targeting to human rDNA loci has higher intrachromosomal recombination efficiency. Gene targeting efficiency in mammalian cells may not be improved by increasing the amount of transfected DNA or the number of copies of the genomic target sequence.11 It can be more sensitive to chromosomal position effects; there is evidence for a position effect on gene targeting in mammalian cells.12 The results of this study illuminate rDNA loci may be homologous recombination hotspots.In this research, the expression of the VEGF165 fusionFig. 3. A: RT-PCR results. M: DNA marker; Lines 1-4: site-specific integration clone; line 5: HT-1080. B : Individual drug-resistant colony expressing fusion protein after transfection under the fluorescence microscope (original magnification×10) C: Western blot analysis of VEGF with anti-human VEGF antibody and anti-GFP antibody. Line 1: site-specific integration clones; Line 2: HT1080 cells.protein in site-specific integration clones were detected successfully by ELISA and Western blot; with an expression quantity of (1000±23) pg·10-6 cells·24 h-1. Human VEFG consists of five different isoforms of 121, 145, 165, 189 and 206 amino acids that are derived from alternative splicing at the transcription level. VEGF165 is the most widely expressed isoform and is a prime angiogenic growth factor for migration of endothelial cells and a mediator of vascular permeability. Moreover, it is more potent than other isoforms in stimulating endothelial cell proliferation. The VEFG165 isoform was selected to construct the targeting vector: pHrneo- VEGF165. In the gene therapy for ischemic limb and myocardial diseases a direct local intramuscular injection was employed in most experimental and clinical studies. Clinical researches also suggest that local therapy may be superior to systemic therapy and gene therapy may have more advantages compared with administration of recombinant protein.13 Because the VEGF165 protein can combine with heparin proteoglycan locating to the endothelial cell surface of ischemic muscle tissues, a small quantity of VEGF165 protein can have an efficient effect.14 A study showed that low gene transfection efficiency and low local VEGF concentrations can achieve meaningful biological effects.15 In successful clinical and experimental studies, levels of circulating angiogenic growth factors were either not measurable or in the picogram range.1, 2In conclusion, in our study VEGF165 has been successfully expressed in a hrDNA locus by the targeting vector: pHrneo-VEGF165. Although the expression of VEGF165 was in the picogram range, the targeting vector, pHrneo-VEGF165, can be used for gene therapy of ischemic limb and myocardial diseases by direct local intramuscular injection. This research will provide new insights into the clinical research of ischemic limb and myocardial diseases.REFERENCES1. Shyu KG, Chang H , Wang BW , Kuan P. Intramuscularvascular endothelial growth factor gene therapy in patientswith chronic critical leg ischemia. Am J Med 2003; 114:85-92.2. Vale PR, Losordo DW, Milliken CE, McDonald MC,Gravelin LM, Curry CM, et al. Randomized, single blind,placebo-controlled pilot study of catheter-based myocardialgene transfer for therapeutic angiogenesis using left ventricular electromechanical mapping in patients withchronic myocardial ischemia. Circulation 2001; 103: 2138-2143.3. Losordo DW, Vale PR, Hendel RC,Milliken CE,FortuinFD,Cummings N, et al. Phase 1/2 placebo controlled, doubleblind, dose-escalating trial of myocardial vascular endothelialgrowth factor 2 gene transfer by catheter delivery in patientswith chronic myocardial ischemia. Circulation 2002; 105:2012-2018.4. Wu X, Li Y, Crise B, Burgess SM. Transcription start regionsin the human genome are favored targets for MLV integration.. Science 2003; 300: 1749-1751.5. Schroder AR, Shinn P, Chen H, Berry C, Ecker JR, BushmanF. HIV-1 integration in the human genome favors activegenes and local hotspots. Cell 2002 ; 110: 521-529.6. Kaiser J. Gene therapy. Seeking the cause of inducedleukemias in X-SCID trial. Science 2003; 299: 495-501.7. Eickbush DG, Luan DD, Eickbush TH. Integration ofBombyx mori R2 sequences into the 28S ribosomal RNAgenes of Drosophila melanogaster. Mol Cell Biol 2000; 20:213-223.8. Szostak JW, Wu R. Insertion of a genetic marker into theribosomal DNA of yeast. Plasmid 1979; 2: 536-554.9. Lorenzi HA, Vazquez MP, Levin MJ. Integration ofexpression vectors into the ribosomal locus of Trypanosomacruzi. Gene 2003; 310: 91-99.10. Brown JP, Wei W, Sedivy JM. Bypass of senescence afterdisruption of p21CIP1/WAF1 gene in normal diploid humanfibroblasts. Science 1997; 277: 831-834 .11. Waldman AS. The search for homology does not limit the rateof extrachromosomal homologous recombination in mammalian cells. Genetics 1994; 136: 597-605.12. Yanez RJ, Porter AC. A chromosomal position effect on genetargeting in human cells. Nucleic Acids Res 2002; 30:4892-4901.13. Yla-Herttuala S, Martin JF. Cardiovascular gene therapy.Lancet 2000; 355: 213-222.14. Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascularpermeability factor/vascular endothelial growth factor, microvascular hyperpermeability and angiogenesis. Am JPathol 1995; 146: 1029-1039.15. Losordo DW, Pickering JG, Takeshita S,Leclerc G,Gal D,WeirL, et al. Use of the rabbit ear artery to serially assess foreignprotein secretion after site specific arterial gene transfer invivo:evidence that anatomic identification of successful genetransfer may underestimate the potential magnitude of transgene expression. Circulation 1994; 89: 785-792.(Received January 18, 2007)Edited by CHEN Li-min。

NOR1基因在前列腺癌细胞和组织中的表达

第43卷第5期2018年5月贵州医科大学学报Vol.43 N o.5J O U R N A L O F G U I Z H O U M E D I C A L U N I V E R S I I'Y2018.5WO/?1基因在前列腺癌细胞和组织中的表达+陈云峰，张宇，刘鹏华，陈乐仲(深圳市宝安区中心医院，广东深圳518102)[摘要]目的：探究基因在前列腺癌细胞和组织中的表达及意义。

方法：89例前列腺癌患者手术切除的配对癌组织、癌旁组织及正常组织标本,Real-time P C R检测worn m R N A表达，记录患者的Gleason分值、肿瘤分期、淋巴结转移、远处转移及术前血清前列腺癌特异性标志物P S A资料;构建过表达AOfll质粒转染的前列腺癌细胞DU-145,CCK-8实验检测细胞的增殖能力，Tmnswell实验检测细胞的侵袭能力，Western blot试验检测细胞中Bcl2、Bax及cleaved-capae-3蛋白表达。

结果：AOfll m R N A表达水平比较，前列腺癌组/<癌旁组/<正常组织（P<0. 05) ;N O R1表达与前列腺癌病理分期及淋巴结转移明显有关（P<0. 05);过表达A O W1的前列腺癌细胞增殖侵袭能力减弱（P<0. 05),Bax和cleaved-caspase-3水平上调、Bcl2水平下调。

结论：N0R1在前列腺癌组织中表达下调，恢复其表达可抑制前列腺癌细胞增殖侵袭能力,其机制可能与Ba x和cleaed-capae-3水平上调和Bcl-2水平下调、前列腺癌病理分级及淋巴结转移有关。

[关键词]前列腺癌；抑癌基因,N0R1;细胞增殖；细胞凋亡；临床分期[中图分类号]R737.2[文献标识码]A[文章编号]1000-2707(2018)05-0546-5D O I：10.19367/j. cnki. 1000-2707. 2018. 05. 011The Expression of NOR1Gene in Prostate Cancer Tissues and CellsCHEN Yunfeng, ZH A N G Yu，LIUPenghua, CHENLezhong{Shenzhen Baoan District Central Hospital, Shenzhen 518102 , Guangdong,China)[Abstract] Objective：To investigate the expression and significance of N0R1 gene in prost cells and tissues. Metliods ： Collecting resected cancerous tissue, precancerous lesions and healthy tissue samples of 89 cases of prostate cancer patients; Real-time PCR was used to detect the expression ofN0R1; the G leason score, staging, lymph node metastasis, distant metastasis and preoperative serumPSA levels were collected. The plasmid was transfected to construct N0R1-overexpressing prostatecancer cells; cell proliferation was detected by CCK-8 assay; Tanswell assay was used to detect cell invasion and Western blot was used to detect protein expression of B cl2, Bax and cleaved-caspase-3.Results ：N0R1 mRNA expression level comparison showed that，prostate cancer tissues < p recancerous tissues < normal tissues ( P< 0. 05 ) . The proliferation and invasion ability of prostate cancer cellsoverexpressing N0R1 were decreased(P <0. 05) . The expressions of Bax and cleaved-caspase-3 protein were increased and the expression of Bcl-2 was downregulated. The expression of N0R1 was associated with the pathological grade of prostate cancer and lymph node metastasis (P <0. 05) . Conc sion：The expression of N0R1 in prostate cancer tissues was down-regulated, recovering its expressioncould inhibit the p roliferation and invasion ability of prostate cancer cells. Its mechanism could be associated with the pathological grade of prostate cancer and lymph node metastasis.[K e y w o r d s] prostate cancer ; N0R1 ; cell proliferation ; apoptosis ; clinical staging1基金项目]深圳市宝安区科技计划项目[深宝科(2017)53号]网络出版时间：018 -05 -1/ 网络出版地址：http://kcms/detai/52. 1164. R.2018051/ 1756.010. html546陈云峰等W0R1基因在前列腺癌细胞和组织中的表达5期前列腺癌是男性最常见的恶性肿瘤，占男性癌症的15% [1]。

Vasculogenic mimicry and its clinical significance in medulloblastoma

©2011 L a n d e s B i o s c i e n c e .D o n o t d i s t r i b u t e .Cancer Biology & Therapy 13:5, 1-8; March 1, 2012; © 2012 Landes BioscienceresearCh paper*Correspondence to: Yi-quan Ke; Email: yiquanke@ Submitted: 10/15/11; Revised: 12/18/11; Accepted: 12/18/11/cbt.13.5.19108IntroductionMedulloblastoma is the most common primary malignantbrain tumor in children and young adults. Over the past three decades, many medulloblastoma patients have successfully been cured by multimodal treatments, including surgery, postsurgi-cal radiotherapy and chemotherapy. However, these treatments also resulted in chronic suffering for patients. Cognitive dysfunc-tion and neurological problems were two of the most common complications. Moreover, evidence indicates that the prognosis for medulloblastoma is still poor in a significant proportion of patients.1 Thus, to develop more effective and harmless treat-ments, we need to explore medulloblastoma in detail.Angiogenesis has been considered to be the most impor-tant way for tumors to sustain growth. Without angiogenesis, tumors cannot grow beyond 2–3 mm 3.2 Current research on vasculogenic mimicry (VM) has reinforced this viewpoint. In 1999, Maniotis et al. reported a new blood supply system inde-pendent of endothelial vessels in malignant melanoma, named VM. VM is defined as a fluid-conducting channels formed by highly invasive and genetically dysregulated melanoma cells. Endothelial cells are not present in VM channels as detected by light microscopy, immunohistochemistry and transmission Vasculogenic mimicry (VM), a process involving the formation of a tubular structure by highly invasive and genetically dysregulated tumor cells, can supplement the function of blood vessels to transport nutrients and oxygen to maintain the growth of tumor cells in many malignant tumors. We aimed to explore the existence of VM and its clinical significance in medulloblastoma in this study. VM was identified in 9 out of 41 (22%) medulloblastoma tissues. Immunohistochemical studies revealed that the presence of VM was associated with the expression of MMp-2, MMp-14, epha2 and laminin 5γ2. Tumor tissues with VM were associated with lower microvessel density (MVD), which was indirect evidence of the blood supply function of VM. survival analysis and log-rank tests showed that patients with VM had shorter overall survival time than those without VM. Multivariate analysis and the Cox proportional hazards model identified VM as independent prognostic factor for overall survival. Our results confirmed the existence of VM for the first time and revealed that VM is a strong independent prognostic factor for survival in patients with medulloblastoma.Vasculogenic mimicry and its clinical significancein medulloblastomashi-yong Wang,1,2 Li Yu,3 Geng-qiang Ling,1,2 sha Xiao,3 Xin-lin sun,1,2 Zhen-hua song,1,2 Yi-jing Liu,1,2 Xiao-dan Jiang,1,2Ying-qian Cai 1,2 and Yi-quan Ke 1,2,*1Department of Neurosurgery; and 3pathology; Zhujiang hospital; 2Institute of Neurosurgery; Key Laboratory on Brain Function repair and regeneration of Guangdong;southern Medical University; Guangzhou, ChinaKey words: medulloblastoma, vasculogenic mimicry, microvessel density, prognosis, angiogenesisAbbreviations: VM, vasculogenic mimicry; MVD, microvessel density; KPS, karnofsky performance scale; D/N, desmoplastic/nodular; MBEN, medulloblastoma with extensive nodularity; LCA, large cell/anaplastic; SI, staining index; PI3K,phosphatidylinositol-3-kinaseT h i s m a n u s c r i p t h a s b e e n p u b l i s h e d o n l i n e , p r i o r t o p r i n t i n g . O n c e t h e i s s u e i s c o m p l e t e a n d p a g e n u m b e r s h a v e b e e n a s s i g n e d , t h e c i t a t i o n w i l l c h a n g e a c c o r d i n g l y .electron microscopy.3 Studies revealed that VM exists in many malignant tumors,4-8 and VM is a prognostic factor for poorer clinical outcome.9-13 Research on angiogenesis inhibitors such as Anginex, TNP-470 and endostatin revealed that they could abrogate new vessels formed by human vascular endothelial cells in vitro. Interestingly, these inhibitors did not affect tumor cell VM formation under the same conditions and even induced the formation of VM.14 Medulloblastoma is abundant in vessels, but results from anti-angiogenic treatments are far from satisfac-tory.15 These data suggest that VM functions as a supplementary blood supply system for the growth of tumors and contributes to the failure of anti-angiogenic treatments aimed at depriving tumors of blood supply.This is the first study demonstrating the presence of VM and its clinical significance in medulloblastoma. These findings will provide additional information that will hopefully lead to improvement in targeted therapies for medulloblastoma.Results Clinical characteristics of the patients. Tables 1 and 2 sum-marize the clinical and pathological characteristics of 41 medul-loblastoma patients.©2011 L a n d e s B i o s c i e n c e .D o n o t d i s t r i b u t e .cases. However some channels had characteristics clearly differ-ent from blood vessels in 9 out of 41 cases. The CD34-negative PAS-positive channels were found in 5 out of 25 classic medul-loblastomas, 2 out of 5 desmoplastic medulloblastomas and 2 out of 4 large cell/anaplastic (LCA) medulloblastomas. The PAS-positive patterns, also described as a fluid-conducting ECM meshwork lined with tumor cells, coexisted with endo-thelial cell-lined blood vessels and had direct connections with them. The PAS-positive patterns extended directly from CD34-positive vessels, branched out into several smaller PAS-positive patterns which interconnected into a cluster of back-to-back looping patterns. At high magnification, translucent red blood cells were found spreading along the PAS-positive pat-terns (Fig. 1A–D ). In the adjacent tissue sections, we stained with eosin after PAS-CD34 dual staining and red blood cells were highlighted. We confirmed red blood cells spreading along PAS-positive patterns. In some regions, the patterns were splayed open, showing hollow channels containing red blood cells (Fig. 1E ). These characteristics strongly supported the belief that the PAS-positive patterns were a part of the medul-loblastoma microcirculation.VM is associated with the expression of MMP-2, MMP-14, EphA2 and laminin 5γ2 in medulloblastoma. The results of immunohistochemical studies for VEG F, MMP-2, MMP-14, EphA2 and laminin 5γ2 were summarized in Figure 2 and Table 3. The expression of MMP-2 (p = 0.001), MMP-14 (p = 0.039), laminin 5γ2 (p = 0.005) and EphA2 (0.036) were signifi-cantly higher in the VM-positive group than in the VM-negative group, whereas the expression of VEGF (p = 0.518) showed no difference between these two groups.No association was found between VM and clinical and pathological data. The presence of VM in medulloblastoma was not associated with age (p = 0.187), gender (p = 0.819), Karnofsky Performance Scale (KPS) score (0.224), hydrocephalus (0.580), histological classification (0.286), tumor location (p = 0.724), tumor size (p = 0.530), tumor metastasis (p = 0.479), and the extent of tumor resection (p = 0.819) (Table 2).VM-positive medulloblastoma has lower microvessel den-sity. We compared the MVD counts between VM positive and VM negative medulloblastoma. The results demonstrated that VM positive medulloblastomas had lower levels of MVD (38.12 ± 5.64) when compared with VM negative medulloblastomas (58.53 ± 11.53), (p = 0.000) (Table 4).VM is a prog nostic factor for shorter survival time in medulloblastoma. We used Kaplan-Meier survival analysis to compare the survival times between VM-positive (total 9, censored 2) and VM-negative patients (total 32, censored 18) or between below-median MVD (total 21, censored 9) and above-median MVD (total 20, censored 11). The results dem-onstrated that the VM-positive group had a shorter survival time (median 30.47 mo, 95% CI, 24.00–36.94 mo) when com-pared with the VM-negative group (median 56.43 mo, 95% CI 46.98–65.89 mo) (p < 0.0001) (Fig. 3). There was no sig-nificant correlation between MVD and overall survival time of the medulloblastoma patients (p = 0.386) (Fig. 4). Univariate analysis showed that survival time was significantly correlatedVM exists in medulloblastoma. We used the CD34 anti-gen as a hallmark to identify endothelial cells, and we usedperiodic acid-Schiff (PAS) staining to highlight the basement membrane of tumor blood vessels and the PAS-positive pat-terns in medulloblastoma tissue sections. Tumor blood vessels positive for CD34 showed brown staining on the luminal sur-face and a PAS-positive reaction in the basement membrane. CD34-positive blood vessels played a dominant role in mostTable 1. Clinical data of medulloblastoma patientsDataMedian age (years)9 (1.5–37)Median MVD counts 51.5Median Kps score 80 (50–90)Median tumor size (cm 3)21.0Median survival time (months)51.2Table 2. The association of clinicopothological data and VM in medulloblastoma patientsnVM +VM -χ2*pGender Male 266200.0530.819Female 15312Age (years)age ≤31239 3.3530.1873< age ≤1520614age >15909KPS score ≤70206141.4760.224≥8021318HydrocephalusNo154110.3070.580Yes26521Histology Classic 30525 2.5050.286Desmoplastic725LCa422Tumor locationmidline 307230.1250.724lateral 1129Tumor size ≤median 224180.3940.530>median 19514Metastasis positive 10370.5000.479Negative 31625Resection Total 266200.0530.819partial15312*pearson χ2 test (asymptotic significance, two-sided).©2011L a n d e s B i o s c i e n c e.D o n o t d i s t r i b u t e.Figure 1. representative microphotographs of endothelial cell-lined blood vessels and VM in medulloblastoma. (a) pas-positive patterns were indirect communication with CD34 positive endothelial cell-lined blood vessels (brown stainings). The pas-positive patterns extended directly fromCD34-positive vessels and branched out into smaller pas-positive patterns that interconnected with a cluster of back-to-back looping patterns (Boxed area). (B) In high magnification field, translucent red blood cells were found spreading along the pas-positive patterns (red arrow). (C) Tumor cells delimited by cross-linking pas-positive patterns. Boxed area was illustrated in oil immersion objective in (D). (D) In some regions, pas-positive patterns showed hollow channels filled with translucent red blood cells (red arrow). (e) patterns in medulloblastoma were stained with CD34 and pas dual staining in combination with eosin staining to highlight red blood cells. pas-positive patterns splayed open and filled with red blood cells, withouthints of endothelial cells in certain areas (red arrow). Tissue stains: CD34 and pas dual staining with hematoxylin counterstaining (a–D) and in combi-nation with eosin (e). Original magnifications, x200 (a), x400 (B), x400 (C), x1,000 (D), x400 (e).©2011 L a n d e s B i o s c i e n c e .D o n o t d i s t r i b u t e .DiscussionThe vascularization of tumors is heterogeneous. Angiogenesis (the tumor vasculature arising from sprouting of endothelial cells from local vessels) and vasculogenesis (vasculature formed by colonization of circulating endothelial progenitor cells from the bone marrow) were once two well-accepted paradigms for thewith VM (p = 0.0001), metastasis (p = 0.044) and KPS score (p = 0.01). Patient age, gender, hydrocephalus, tumor histo-logical classification, tumor size, tumor location and extent of resection were not significant variables. Multivariate analysis and the Cox proportional hazards model identified VM and metastasis as independent prognostic factors for overall sur-vival time.Figure 2. Immunohistochemical studies of the expression of VeGF, MMp-2, MMp-14, epha2 and laminin 5γ2 in medulloblastoma. (a, C, e, G and I) arerepresentative micrographs from VM-positive tissue sections. (B, D, F, h and J) are representative micrographs from VM-negative tissue sections.(a and B) expression of VeGF in medulloblastoma. (C and D) expression of MMp-2 in medulloblastoma. (e and F) expression of MMp-14 in medulloblas-toma. (G and h) expression of epha2 in medulloblastoma. (h and I) expression of laminin 5γ2 in medulloblastoma. Original magnifications, x400 (a–J).©2011 L a n d e s B i o s c i e n c e .D o n o t d i s t r i b u t e .expression of MMP-2, MMP-14, EphA2 and laminin 5γ2 in VM-positive tissues compared with VM-negative tissues, espe-cially in those VM lesions. These components play significant roles in the formation of VM. Studies in melanoma indicated that cooperative interactions between laminin 5γ2-chain and MMP-2 and MMP-14 are required for the formation of VM. EphA2 can activate phosphatidylinositol-3-kinase (PI3K), which regulates the activity of MMP-14. Activated MMP-14 leads to the activation of pro-MMP-2 to an active MMP2 proteinase. Active MMP-2 can cleave laminin 5γ2-chain into promigratory γ2' and γ2x fragments, which can lead to the formation of VM.26,27 Our formation of tumor vascularization. The blood vessels formed by angiogenesis and vasculogenesis are both initiated and regulated by angiogenic factors secreted by tumor cells.16 However, theseobservations have been challenged over the last decade by thediscovery of VM in a series of malignant tumors. Until now, the underlying molecular mechanisms for VM were not completely understood. Previous research on melanoma, breast cancer, ovarian cancer suggest that the plasticity of cancer cells enablesthem to mimic the phenotypes and functions of endothelial cells, thus leading to the formation of patterned matrix type of VM-patterned networks of interconnected loops of PAS-positiveextracellular matrix that may be solid or hollow, with no involve-ment of endothelial cells.17-19 The plasticity of highly malignanttumor cells has some similarities to the stemness of cancer stemcells. Recently, reports reveal that glioblastoma stem-like cells (CSC) can differentiate into functional endothelial cells, which harbor the same genomic alterations as cancer cells.20,21 Andthere is evidence suggesting that glioblastoma stem-like cells are capable of forming blood vessels de novo-tubular type of VM.5 Together, these observations strongly suggest that the plasticity of highly malignant tumor cells and cancer stem-like cell may mimic the role of normal stem cells and facilitate the formation of vascular network to sustain the blood supply of fast-growing tumor.22Morphologically, VM channels are patterned networks of solid or hollow interconnected loops of PAS-positive extracel-lular matrix, lined externally by tumor cells. Functionally, they interconnect with endothelial blood vessels and conduct plasma and red blood cells. Previous studies in mela-noma provided considerable evidence that the looping patterned matrix conducts fluid in vitro and in vivo. Melanoma cells cultured in three-dimensional matrix gel formed looping patterns which can transmit fluid after direct microinjection and by passive absorption.23 In a xenograft mouse model of uveal melanoma, intravenous tracer material can perfuse to a looping patterned matrix.24 Further studies in patients with uveal melanoma using laser scanning confocal microscopy revealed similar results.25 Based on three morphological mani-festations, we believe that the PAS-positive patterns in medulloblastoma are a part of the tumor microcirculation, but not blood leaks or hemorrhage. First, the patterned matrix was in direct communication with blood ves-sels. Second, in certain regions, these patterns branched out into smaller patterns with hol-low channels filled with red blood cells. Third, using the CD34 and PAS dual staining in combination with eosin staining to highlight the red blood cells in tissue sections, we found that red blood cells spread along the PAS-positive patterns.In this article, immunohistochemical studies revealed significant increases in the Table 3. expression of VeGF, MMp-2, MMp-14, epha2 and laminin 5γ2 in medulloblastoma tissuesVM +VM -Z p value VeGF 65.20 ± 36.2071.94 ± 54.66-0.6460.518MMp-2112.56 ± 56.7760.48 ± 60.07-3.1970.001MMp-14115.16 ± 85.5573.47 ± 53.64-2.0040.039epha252.07 ± 18.3933.78 ± 20.88-2.0950.036Laminin 5γ2129.91 ± 95.8459.03 ± 63.11-2.8030.005Table 4. Correlation of MVD with VM in medulloblastoma N MVD (mean ± SD)Z p VM +938.12 ± 5.64VM -3258.53 ± 11.53-4.220.000Figure 3. The presence of VM in medulloblastoma was associated with shorter survival time by Kaplan-Meier survival analysis and log-rank test (VM-positive 9, censored 2 vs. VM-neget-ive 32, censored 18).©2011 L a n d e s B i s c i e n c e .D o n o t d i s t r i b u t e .metastasis,22 there was no association between VM and metastasis in our study. Our obser-vation needs to be further studied in larger amount of samples. The MVD counts in the VM-positive group were significantly less than in the VM-negative group, which is indirect evidence of the blood supply function of VM. A previous study revealed that MVD has no prognostic significance in medulloblastoma.29 Our observations in medulloblastoma lead us to the same conclusion.Observations in a series of malignant tumors reveal that VM might serve as an unfavorable prognostic factor. However, there has been no research on the prognostic signifi-cance of VM in medulloblastoma prior to our study. Although VM had no association with patients’ clinicopathological characteristics in this study, Kaplan-Meier survival analy-ses and log-rank tests revealed that medullo-blastomas with VM have a poorer clinical outcome compared with those without VM. Univariate analysis and multivariate analysis showed that VM is an independent prognos-tic factor for overall survival time in patientswith medulloblastoma. Our observation in medulloblastoma is consistent with previous studies. It is noteworthy that while VM cor-relates with the MVD, MVD does not cor-relate with overall survival, yet VM does. It is not surprising that, although we found no correlation between VM and pathological classifications or between VM and metastasis which was consid-ered as inconclusive results in this study, VM is indeed associated with more malignant biological behaviors such as invasiveness and metastasis that directly affect patient clinical outcome.3 Moreover, currently widespread used anti-angiogenic drugs may have no effect on VM,14 even inducing VM formation when blood vessels are destroyed leaving a hypoxic environment.30 Therefore,newly developed drugs based on anti-angiogenic strategies must take both anti-angiogenic and anti-VM treatment into serious consideration.In conclusion, this pilot study provided direct evidence that VM, a supplementary microcirculation for angiogenesis, exists in medulloblastoma and is an adverse predictor of clinical outcome.Materials and Methods Tissue samples. A total of 41 paraffin-embedded medullo-blastoma tissues between 1999 and 2010 were obtained from the Department of Pathology, Zhujiang Hospital, Southern Medical University. All tissue samples were collected from patients who did not undergo therapy before the surgical opera-tion to remove the tumor. Tumor sections were reviewed by two neuropathologists to verify the diagnosis of medulloblas-toma in accordance with the 2007 World Health Organizationresults provide evidence that VM may be associated with the deg-radation and remodeling of the extracellular matrix in medul-loblastoma. Research on VEGF reveals that it is a stimulator of endothelial cell proliferation, and VEGF can induce the forma-tion of VM through a VEGFR-1dependent manner, increasing the expression of VM associated proteins in melanoma.28 In our research, the expression of VEGF had no significant correlation with VM in medulloblastoma. It is possible that VM in medul-loblastoma might be regulated through a pathway distinct from VEGF.We have confirmed the existence of VM in medulloblas-toma. Moreover, we explored whether the presence of VM has any association with clinicopathological data and its prognosticsignificance in medulloblastoma. In this retrospective study of 41 patients with medulloblastoma, we found that VM had no association with clinicopathological characteristics. Notably VM had no association with histological classification, which wasinconsistent with previous studies in glioma, renal cell carcinomaand mesothelial sarcoma.9,10,12Among all the subtypes of medul-loblastoma, the LCA variant is notable for its high aggressive-ness and relatively poorer clinical outcome.1However, it might be unconvincing to draw the above conclusion as there are only four examples of the LCA subtype in this study. The true asso-ciation has yet to be conclusively determined. Metastases in this study were found in 10 patients: 8 with spinal cord metas-tases and 2 with extracranial metastases. Although the genera-tion of VM by highly aggressive tumor cells facilitates tumor Figure 4. Comparison of Kaplan-Meier survival curves (log-rank test) in patients with medulloblastoma according to patients with MVD values below the group median (total 21,censored 9) vs. those with values above the group median (total 20, censored 11).©2011 L a n d e s B i o s c i e n c e .D o n o t d i s t r i b u t e .percentage of positively stained cells in 10 visual fields of eachsection was converted into a score as follows: 0 for <10% posi-tive cells, 1 for <25% positive cells, 2 for <50% positive cells, 3 for >50% positive cells. The score of intensity of each section was graded as follows: Strong intensity staining was scored as 3, moderate as 2, weak as 1 and negative as 0. The staining index was determined by multiplying the average percentage of positive cells and the score of intensity. The assessment of immunohisto-chemistry results was completed by two independent investiga-tors blinded to the clinicopathological data. Discrepancies were resolved by consensus.9Microvessel density. Microvessel density counts were com-pleted according to the method described by Foote et al. This portion of the study was completed by two additional inde-pendent observers unaware of the clinical outcome of patients. Tumor sections were examined under 200x magnification, and the average number of microvessels in 10 randomly selected visual fields represented the MVD of the tumor. Any endothe-lial cell or endothelial cell cluster positive for CD34 and clearly separate from the adjacent cluster was considered to be a single countable microvessel. The average MVD for the 41 specimens was calculated and patients were grouped according to whether their MVD was lower (below-median MVD) or higher (above-median MVD) than median. The overall survival times for these two groups were compared, and the association between VM and MVD was determined.Statistical analysis. Statistical analyses were performed using SPSS 13.0 for Windows (SPSS Inc.). A p value of less than 0.05 was defined as statistically significant. The differential expres-sion levels of VEGF, MMP-2, MMP-14, laminin 5γ2, EphA2 in the VM-positive and VM-negative groups were determined by the Mann-Whitney test. MVD counts in the VM-positive and VM-negative groups were also compared by the Mann-Whitney test. Associations between VM and clinical and pathological data were determined by the Pearson’s χ2 test. The survival analysis was calculated by the Kaplan-Meier method and the differ-ences between groups were compared using the log-rank test. Univariate analysis was performed to identify prognostic vari-ables for overall survival. Multivariate analysis was performed to identify the independent prognostic factors for survival using the Cox regression hazard model. Data on surviving patients and patients without follow-up information were considered censored data in the analysis.Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.AcknowledgementsWe express our gratitude to Professor Shengli An (Department of Biostatistics, Southern Medical University) for the analysis of statistical data and Mr. Xuan Lu (Department of Pathology, Zhujiang Hospital, Southern Medical University) for the sec-tioning of tissues. This project was supported by grants from the National Science Foundation of China (No. 30901174), and from the Science and Technology Planning Project of Guangdong (No. 2008B030301152).(WHO) classification of central nervous system (CNS) tumors. According to the 2007 WHO classification of CNS tumors, medulloblastomas are separated into the classic tumor and four main histological subtypes:desmoplastic/nodular (D/N), medulloblastoma with extensive nodularity (MBEN), ana-plastic medulloblastoma and large cell medulloblastoma. D/N medulloblastoma and MBEN share fundamental clinical and pathological features as do anaplastic medulloblastoma and large cell medulloblastoma.1,31 We divided the 41 patients into three groups: classic medulloblastoma, desmoplastic medullo-blastoma that contained D/N and MBEN medulloblastomas and LCA medulloblastoma that contained anaplastic medullo-blastoma and large cell medulloblastoma. Detailed clinical and pathological data were collected for all samples, including age, gender, KPS score, hydrocephalus, histological classification, size, location, metastasis, total or partial resection. Research Ethics Committee approval has been obtained for the use of all human tissues in this study.CD34-PAS dual staining. For CD34-PAS dual staining par-affin sections were cut at 5 μm. Slides were deparaffinized twice with xylene and subsequently hydrated against a concentration gradient in ethanol solutions. Antigen retrieval was performed using citrate buffer (pH 6.0) at 96°C for 20 min. For demon-stration of endothelial cells in medulloblastoma, the slides were incubated with mouse monoclonal anti-human CD34 antibody (Santa Cruz Biotechnology, catalog: sc-52312, dilution 1:100) at 4°C over night. After washing with PBS, the slides were treated with PowerVision Two-Step Histostaining Reagent (Zhongshan G oldenbridge Biotechnology, catalog: PV-6002) for 30 min at 37°C. The chromogen used to highlight a positive reaction was 3,3'-diaminebenzidine tetrahydrochloride (DAB), resulting in a brown product. To highlight the matrix-associated vascular channels of medulloblastoma, slides were stained following the PAS staining procedures before counterstaining with Mayer’s hematoxylin. Finally, the slides were covered with a permanent mounting medium. To highlight the red blood cells in PAS posi-tive patterns, the adjacent sections were stained with eosin after the above procedures.Immunohistochemistry. To determine the expression of VEGR, MMP-2, MMP-14, EphA2 and laminin 5γ2 in medul-loblastoma by immunohistochemistry, tissue sections were pre-pared as previously described. Slides were then incubated over night at 4°C with a monoclonal antibody against VEGF (Santa Cruz Biotechnology, catalog: sc-80437, dilution 1:50); EphA2 (Millipore, catalog: 05-480, dilution 1:100), MMP-2 (Abcam, catalog: ab2462, dilution 1:100), MMP-14 (Abcam, catalog: ab53712, dilution 1:100), or a polyclonal antibody against lam-inin 5γ2 (Abcam, catalog: ab96327, dilution 1:100). Appropriate positive and negative controls for each antibody were included. The staining index (SI) was used to evaluate the expression level of each marker based on the varying degrees of staining inten-sity and percentage of positively stained cells in the specimens. Therefore, a combined intensity and percentage positive scoring method was used. Ten visual fields of each tissue section were selected randomly under the microscope at 400x magnification, and 100 cells in each visual field were counted. The average。

Expression of vasorin (Vasn) during embryonic development of the mouse

Expression of vasorin (Vasn )during embryonic development of the mouseA.Michaela Krautzberger a ,1,Barbara Kosiol a ,Manuela Scholze a ,b ,Heinrich Schrewe a ,b ,⇑a Department of Developmental Genetics,Max Planck Institute for Molecular Genetics,Ihnestrasse 63-73,14195Berlin,GermanybInstitute for Medical Genetics,Charité-University Medicine Berlin,Campus Benjamin Franklin,Hindenburgdamm 30,12203Berlin,Germanya r t i c l e i n f o Article history:Received 19January 2012Accepted 28February 2012Available online 8March 2012Keywords:Vasorin Vasn Slitl2AtiaTransmembrane protein Mouse embryo DevelopmentExpression patternWhole mount in situ hybridization lacZ knock-inVascular smooth muscle cells Skeletal system Kidney Lunga b s t r a c tThe murine vasorin (Vasn )gene,initially known as Slit-like 2,encodes a transmembrane protein that shares structural similarities with the eponymous Slit proteins.However,whether it also shares func-tional similarities with these large secreted proteins remains to be elucidated.Here,we report expression of Vasn during embryonic and fetal development of the mouse using whole-mount in situ hybridization (WISH)and histochemical detection of b -galactosidase expressed from a targeted Vasn lacZ knock-in parison of whole-mount staining patterns of both approaches showed identical expression domains,conﬁrming that Vasn promoter-driven b -galactosidase expression faithfully reﬂects endogenous Vasn expression.Vasn is highly expressed in vascular smooth muscle cells (hence the name),a ﬁnding consis-tent with a previous report on its human homolog VASN ,whose extracellular domain was shown to func-tion as a TGF-b trap (Ikeda et al.,2004).Most striking,however,is Vasn ’s prominent expression in the developing skeletal system,starting as early as the ﬁrst mesenchymal condensations appear.Moreover,distinct expression domains outside the bones,e.g.,in the developing kidneys and lungs,suggest further roles for this gene in the mouse.Recently,it was shown that mitochondria-localized Vasn protects cells from TNF a -and hypoxia-induced apoptosis,and partial deletion of the Vasn coding sequence leads to increased sensitivity of hepatocytes to TNF a -induced apoptosis (Choksi et al.,2011).By providing a ﬁrst comprehensive analysis of the Vasn expression pattern during mouse embryonic development,our study will help to further elucidate its biological functions.Ó2012Elsevier B.V.All rights reserved.1.Results and discussionVasorin (Vasn )was ﬁrst isolated from a mouse kidney cDNA li-brary.The locus consists of two exons spanning over a region of approximately 11kb on mouse chromosome 16.Its coding region is entirely located on exon 2,which translates into a typical sin-gle-pass type I transmembrane protein with a calculated molecular weight of 72kDa.Based on the combination of leucine-rich repeats (LRRs)and an epidermal growth factor (EGF)-like motif –the deﬁn-ing feature of the Slit family of proteins –the gene was originally named Slit-like 2(Slitl2).It is as yet unknown whether this protein shares overlapping functions with the eponymous Slit proteins,which are best known for their role in axon guidance (Ypsilanti et al.,2010).Vasn homologs have been identiﬁed in numerous other species.In zebraﬁsh,vasn is expressed in embryonic midline structures of the central nervous system as well as in the vascular system (Chen et al.,2005).The human homolog VASN shares 84%sequence identity with the murine protein.It has been demon-strated that VASN’s extracellular domain negatively modulates TGF-b signaling by sequestering the ligand in a complex,thereby preventing ligand–receptor interactions and subsequent down-stream signaling (Ikeda et al.,2004;Malapeira et al.,2010).Proteo-mic analyses revealed VASN as potential biomarker in kidney disease and cancer (Caccia et al.,2011;Moon et al.,2011).In mice,Vasn expression increases gradually during embryonic develop-ment,as evidenced by Northern blot analysis,and in situ hybridiza-tion showed expression in developing and adult aortic vascular smooth muscle cells (Ikeda et al.,2004).Recently,it was demon-strated that Vasn protects cells against TNF a -and hypoxia-induced apoptosis through regulation of the mitochondrial antioxidant thi-oredoxin-2(TRX2),and that mice with a partial deletion of the Vasn coding sequence show increased susceptibility to apoptosis in a model of TNF a -induced hepatitis (Choksi et al.,2011).To delineate in detail the tissues that may require Vasn during mouse development,we assayed endogenous Vasn mRNA expres-sion using whole-mount in situ hybridization (WISH)from embry-onic day 8.5(E8.5)to E11.5.In addition,we generated a Vasn lacZ knock-in reporter mouse strain.In these mice,the Vasn coding se-quence is replaced by the b -galactosidase (lacZ )open reading frame fused to a nuclear localization signal on one allele,thereby placing lacZ under transcriptional control of the endogenous Vasn regula-1567-133X/$-see front matter Ó2012Elsevier B.V.All rights reserved./10.1016/j.gep.2012.02.003⇑Corresponding author.Tel.:+493084131302;fax:+493084131216.E-mail address:schrewe@molgen.mpg.de (H.Schrewe).1Present address:Department of Stem Cell and Regenerative Biology,Harvard University,7Divinity Avenue,Cambridge,MA 02138,USA.tory elements(Fig.1A).Correct integration of the targeting frag-ment in embryonic stem cells was conﬁrmed by PCR(Fig.1B). Cre-mediated recombination led to excision of the selection cas-sette,and heterozygous Vasn lacZ/+mice were identiﬁed by PCR (Fig.1C).Histochemical detection of b-galactosidase protein expres-sion from the targeted allele in Vasn lacZ/+embryos produced an expression proﬁle consistent with the WISH analysis of endogenous Vasn expression from E8.5to E11.5and thus was further employed for detailed analysis of Vasn expression domains throughout embry-onic development.1.1.Whole-mount expression analysis of Vasnparison of WISH and X-gal staining patterns from E8.5to E11.5In the E8.5embryo,endogenous Vasn mRNA and Vasn pro-moter-driven reporter protein expression is detectable in the hind-brain and the midline of the neural tube.In addition,the lateral mesoderm and the caudal extremity exhibit staining(Fig.2A and E).At E9.5,expression is prominent in the rhombic lip,a restricted region adjacent to the hindbrain roof plate.Cross sections show expression in the roof plate and,in addition,reveal staining in theﬂoor plate.Moreover,theﬁrst branchial arches,the septum transversum,the emerging forelimb buds,and the lining of the coelomic cavity reveal staining(Fig.2B–B’’and F–F’’).Whole-mount specimens at E10.5again show identical expression do-1.1.2.X-gal staining patterns from E12.5to E15.5Despite the reduced substrate penetration efﬁciency owing to an increasing skin thickness,X-gal-stained Vasn lacZ/+embryos from stages E12.5to E15.5all display striking reporter gene activity throughout the developing skeletal system.Here,signal is detected in the axial as well as the appendicular skeleton,e.g.,in the verte-bral bodies and the long bones of the limbs,respectively(Fig.2I–L). As shown for E9.5,wild-type littermates at all stages examined do not exhibit any X-gal staining(Fig.2M).1.2.Histological expression analysis of VasnTo identify Vasn expression domains on a cellular level,we have analyzed sagittal sections at early and middle stages of both organ-ogenesis(E10.5and E12.0,respectively)and fetal mouse develop-ment(E14.5and E17.5,respectively)for b-galactosidase activity in heterozygous Vasn lacZ/+embryos.1.2.1.E10.5Section analysis conﬁrms lacZ activity throughout the cephalic, branchial arch,and trunk mesenchyme(Fig.3A).In the developing heart,reporter signal is mainly found in the myoepicardial layer of the outﬂow tract,while lacZ activity is barely detected in the cells of the common atrial and bulbus cordis region(Fig.3B).Inferior to the common atrial chamber,the lung buds become evident at this stage of embryogenesis;b-galactosidase activity is not detectable in the epithelium of the developing main bronchi(Fig.3C).Epithe-lial cells budding off from theﬂoor of the primitive pharynx to form the thyroid primordium are devoid of X-gal staining (Fig.3D).In the trunk region,strong signal is detected in the sep-tum transversum,whereas the invading hepatoblasts appear un-stained.In the adjacent cystic primordium,staining is most dense in the stalk cells(Fig.3E).Reporter protein expression is notable in epithelial cells lining the mid-and hindgut(Fig.3F). Similarly,Vasn promoter activity is evidenced by X-gal staining in mesonephric tubular cells(Fig.3G).1.2.2.E12.0By E12.0,substantial changes have occurred with regard to the development of the future spine.While lacZ activity is barely de-tected in the newly formed dorsal root ganglia,the intervening, caudal condensed portions of the sclerotomes are clearly marked by b-galactosidase protein expression(Fig.4A and B).Intense sig-nal is also observed in the parietal and visceral mesothelial cells lining the coelomic cavities and internal organs,respectively.In the developing heart,for example,X-gal staining is most intense in the pericardium,but rather sparse in the ventricular trabeculat-ed muscle and atrio-ventricular cushion tissue(Fig.4A and C). Likewise,staining mostly localizes to the cells surrounding the he-patic parenchymal tissue,in which only scattered nuclear lacZ activity is observed(Fig.4C).The cells of the pancreatic primordia, on the other hand,display homogenous b-galactosidase activity (Fig.4D).The urogenital system demonstrates a differential stain-ing proﬁle with sparse signal in the gonadal primordium at its medial aspect,but evenly distributed signal throughout the meso-nephric tissue with its degenerating tubules at its lateral aspect (Fig.4E).Homogenous nuclear b-galactosidase activity is also ob-served throughout the condensed metanephric mesenchyme and the ureteric bud stalk located at the caudal part of the urogenital ridge(Fig.4F).Moreover,the wall of the dorsal aorta shows intense staining(Fig.4G).1.2.3.E14.5As stated for the whole-mount analysis,X-gal staining is partic-ularly prominent throughout all parts of the developing skeletal system from this stage onwards,including areas of both intramem-1.Targeting of the Vasn gene to produce a Vasn lacZ reporter strain(A)Targetingscheme showing wild-type allele and targeting construct used to produceVasn neo-lacZ allele by homologous recombination.The Vasn lacZ allele was generatedsubsequent Cre-mediated excision of the selection cassette.(B)Results of long-range PCR conﬁrming correct recombination of the targeting construct in ES cells.External forward/reverse neo primer and lacZ forward/external reverse primersused for5’and3’end,respectively,are indicated as arrows in(A).(C)Resultsthree-primer PCR to identify heterozygous Vasn lacZ/+embryos.Primers useddiscrimination between the wild-type and Vasn lacZ allele are indicated as arrow-heads in(A).168 A.M.Krautzberger et al./Gene Expression Patterns12(2012)167–171branous and endochondral ossiﬁcation(Fig.5A).As opposed to the previous stage,dispersed lacZ activity is now also detectable in the dorsal root ganglia and–to a lesser extent–also within the cells of the developing brain and spinal cord,while being virtually absent in the pituitary gland primordium(Fig.5B and C).In the two lobes of the thymus gland,quite substantial structures at this stage,lacZ activity is mostly localized in the capsule and the septa emerging from it(Fig.5D).By E14.5,the lungs contain numerous primary,sec-ondary,and tertiary bronchi.While b-galactosidase activity is sparse in bronchial epithelial cells,X-gal staining persists in the pul-monary mesenchymal tissue and is most dense within vessel walls (Fig.5E).The increasing degree of differentiation with the advance-ment of development can also be appreciated with regard to the regionalized staining in the kidney.In the primitive nephrons, marked Vasn promoter activity is observed in the proximal parts of the S-shaped bodies.In the adjacent adrenal glands,reporter pro-analysis of Vasn(A–H)Comparison of whole-mount in situ hybridization(A–D)and X-gal staining(E–H)patterns protein expression is notable in the hindbrain region and the midline of the neural tube(arrow);also,theand E).By E9.5,expression is detected in the rhombic lip(arrowhead in B and F;B0and F0show dorsal view), transversum(black arrow),and in the developing forelimb bud(white arrow)(B and F).Cross sections reveal staining F’’).At E10.5,staining is seen throughout the head and trunk mesenchyme;it remains prominent in evident in the developing hindlimb buds(white arrow)in addition to the forelimb buds(fb)(C and G).In E11.5whole marked by Vasn mRNA and lacZ protein expression(D and H).(I-L)X-gal staining patterns from E12.5to E15.5.axial and appendicular skeletal system.(M)Wild-type littermates(E9.5shown)do not exhibit X-gal staining.Vasn lacZ expression at E10.5(A)Section analysis shows widespread reporter activity throughout the head and most prominent in the outﬂow tract(ot),but weak in the common atrial chamber(ac)and the bulbus cordisinvading lacZ-positive mesenchyme.(D)Cells of the thyroid primordium(dashed circle)show no X-gal staining.negligible in invading hepatoblasts(hb).In the cystic primordium(cy),signal is predominantly located in the stalk lining the midgut(arrow).(G)Mesonephric tubular cells(arrows)display nuclear lacZ activity.(Magniﬁcationtein expression is barely detected in both the mesoderm-derived cortex and the ectoderm-derived medulla(Fig.5F).Histological examination of a male embryo reveals scattered b-galactosidase activity in the cells of the primitive seminiferous tubules,while in-areas of ossiﬁcation have developed,e.g.,within the shaft region of the long bones,where intense staining can be observed (Fig.6B).Dramatic changes are also observed with regard to the appearance of the lungs compared to the previous stage,notablyVasn lacZ expression at E12.0(A and B)LacZ staining is barely detected in newly formed dorsal root ganglia(asterisks), and C)Intense signal is detected in parietal and visceral mesothelial cells.In the developing heart(ventricularexpression is highest in the pericardial and hepatic capsule cells,respectively.(D)Homogenous b-galactosidase (arrow).(E)In contrast to the degenerating mesonephric tubules(arrow),X-gal staining is barely detectable mesenchyme(dashed area)and ureteric bud stalk(ub)both exhibit nuclear lacZ activity.(G)The wall of the dorsal objective:10Âin C;20Âin B,D,E and G;40Âin F).Vasn lacZ expression at E14.5(A)At E14.5,b-galactosidase activity is most prominent in the developing skeletal is now visible in the dorsal root ganglia(asterisks),(C)while the pituitary primordium is devoid of X-galin the thymus gland.(E)In the developing lung,expression is most dense within vessel walls(arrowheads), (asterisks).(F)In the developing kidney,lacZ mostly localizes to the proximal part of the S-shaped bodies(arrowheads).(G)Dense X-gal staining is seen in the mesothelial and interstitial cells of the testis,whereas the primitivestaining is strongest in the cells forming the capsule of the ovary(inset).(Magniﬁcation factor of objective:Vasn lacZ expression at E17.5(A and B)Similar to the previous stage,reporter protein expression is most notablee.g.,within the shaft region of the femur(arrows in B).(C)Generalized weak signal is detected throughout (bronchus,b).In comparison to venous vessels(v),vascular smooth muscle cell-rich arterial vessels(a)show intense to the gastric body region–exhibits marked lacZ activity.(E)Staining is strongest in the submucosal layer predominantely found in the capsule and the arterial tree arising from the hilus(arrow).(G)Staining signal is the pancreas,but homogenous in the pancreatic duct system(arrow).Asterisk indicates arterial vessel,5Âin B–D;10Âin E and F;20Âin G).also observed in the lower intestinal tract.Cross-sectioning of the small intestine reveals that lacZ mostly localizes to the submucosal layer(Fig.6E).In the spleen,strong signal is evident in the capsule as well as in the arterial tree arising from the hilus,whereas the parenchymal tissue itself only exhibits sparse staining(Fig.6F). Similarly,the arterial vessels in the pancreas exhibit strong b-galactosidase activity.Homogenous nuclear lacZ activity is seen in the pancreatic duct system,while staining is sparse in both the endocrine and exocrine components,i.e.,in the islets of Langer-hans and the acinar cells,respectively(Fig.6G).In this report,we haveﬁrst characterized in detail the expres-sion pattern of Vasn during mouse embryonic development.With various distinct expression domains,our results indicate important roles for Vasn e.g.,in the developing skeletal system,the kidneys, and the lungs,and should thus help to further elucidate its biolog-ical functions.2.Experimental procedures2.1.Whole-mount in situ hybridizationFor the generation of the probe,the plasmid pBluescript II KS-Vasn containing a1.8kb-long fragment of the murine Vasn cDNA sequence(accession number AJ458938)was linearized with EcoR I, and anti-sense,digoxygenin(DIG)-labeled probe was obtained with the DIG RNA Labeling Kit(Roche)using T7polymerase.In or-der to reduce probe length,it was incubated in hydrolysis buffer (40mM NaHCO3,75mM Na2CO3,pH10.2)for4min at60°C. Whole-mount in situ hybridization was performed according to the protocol of the‘‘molecular anatomy of the mouse embryo pro-ject’’(mamep)which is available online(http://mamep.mol-gen.mpg.de/protocol.html).2.2.Generation of Vasn lacZ miceThe Vasn lacZ knock-in allele was generated by targeted homolo-gous recombination in G4embryonic stem cells(George et al., 2007),thereby replacing the Vasn coding sequence on exon2with the b-galactosidase(lacZ)ORF fused to a nuclear localization signal.A loxP-ﬂanked PGKneo cassette was inserted upstream to select for targeted clones.Correct integration was assessed using the Expand Long Template PCR System(Roche)according to the protocol of the European Conditional Mouse Mutagenesis Program(EUCOMM; )with primer pairs spanning the Vasn lo-cus-targeting vector junctions on both sides.For the5’junction,a forward primer binding to the Vasn intronic sequence(5’-AGGTG-GAGTTTGTGGTCTGG-3’)and a reverse primer binding to the neo cassette(5’-TGGGAAGACAATAGCAGGCATGC-3’)were used.For the3’junction,a forward primer binding to the lacZ ORF(5’-CAC-ATGGCTGAATATCGACGG-3’)and a reverse primer binding to the3’downstream external sequence(5’-ACCTGCCTCACATTTGTTCC-3’) were selected.Homologously recombined ES cells were aggregated with C57Bl/6recipient morulae to obtain chimeras.After germline transmission had been established,mice carrying a Vasn neo-lacZ al-lele were crossed with CMV-Cre transgenic mice(Schwenk et al., 1995)to remove the selection cassette.Mice carrying a Vasn lacZ al-lele were bred with wild-type NMRI mice,and resulting offspring were genotyped with a three-primer PCR to discriminate between wild-type and heterozygous embryos using wild-type forward primers binding to the Vasn exon2(5’-GGCAACTTCTACAGCT-CAGG-3’)and the lacZ ORF(5’-CACATGGCTGAATATCGACGG-3’) for the wild-type and Vasn lacZ allele,respectively,and a common reverse primer binding to the Vasn3’UTR region(5’-AGAT-GAGACCCAGCCCAGAG-3’).2.3.Detection of lacZ activityFor detection of b-galactosidase activity in whole mounts,em-bryos were dissected in cold PBS,ﬁxed in4%PFA/PBS for1h at 4°C,rinsed three times with rinse buffer(5mM EGTA,0.01% deoxycholate,0.02%NP40,2mM MgCl2,in PBS)for15min at RT, and immersed in staining buffer(5mM K3[Fe(CN)6],5mM K4[Fe(CN)6],1mg/ml X-gal,in rinse buffer)until an appropriate staining intensity was obtained.For section analysis,embryos up to E11.5were stained overnight as whole mounts,embedded in parafﬁn,and sectioned.Older embryos were embedded in Tissue-TekÒOCT compound(Ted Pella)and frozen on a metal block cooled with dry ice and ethanol.Cryostat sections(14–20l m)wereﬁxed on ice in1%PFA/PBS for10min,washed twice in cold2mM MgCl2/ PBS for10min,rinsed twice in cold rinse buffer for10min,and incubated overnight in staining buffer at37°C.All sections were counterstained with eosin and mounted in EntellanÒ(Merck).AcknowledgementsWe thank Karol Macura for ES cell-morula aggregation,Sonja Banko and Mirjam Peetz for expert animal caretaking,and Chris Bunce,Alexandra Farrall,Pedro Rocha,and Lars Wittler for critical comments on the manuscript.A.M.K.thanks Ralf Spörle for sharing his knowledge of mouse development.ReferencesCaccia,D.,Zanetti Domingues,L.,Micciche,F.,De Bortoli,M.,Carniti,C.,Mondellini, P.,Bongarzone,I.,2011.Secretome compartment is a valuable source of biomarkers for cancer-relevant pathways.J.Proteome Res.10,4196–4207. Chen,L.,Yao,J.H.,Zhang,S.H.,Wang,L.,Song,H.D.,Xue,J.L.,2005.Slit-like2,a novel zebraﬁsh slit homologue that might involve in zebraﬁsh central neural and vascular mun.336,364–371. Choksi,S.,Lin,Y.,Pobezinskaya,Y.,Chen,L.,Park,C.,Morgan,M.,Li,T.,Jitkaew,S., Cao,X.,Kim,Y.S.,Kim,H.S.,Levitt,P.,Shih,G.,Birre,M.,Deng,C.X.,Liu,Z.G., 2011.A HIF-1target,ATIA,protects cells from apoptosis by modulating the mitochondrial thioredoxin,TRX2.Mol.Cell.42,597–609.George,S.H.,Gertsenstein,M.,Vintersten,K.,Korets-Smith,E.,Murphy,J.,Stevens, M.E.,Haigh,J.J.,Nagy,A.,2007.Developmental and adult phenotyping directly from mutant embryonic stem A104,4455–4460. Ikeda,Y.,Imai,Y.,Kumagai,H.,Nosaka,T.,Morikawa,Y.,Hisaoka,T.,Manabe,I., Maemura,K.,Nakaoka,T.,Imamura,T.,Miyazono,K.,Komuro,I.,Nagai,R., Kitamura,T.,2004.Vasorin,a transforming growth factor beta-binding protein expressed in vascular smooth muscle cells,modulates the arterial response to injury in A101,10732–10737.Malapeira,J.,Esselens, C.,Bech-Serra,J.J.,Canals, F.,Arribas,J.,2010.ADAM17 (TACE)regulates TGFbeta signaling through the cleavage of vasorin.Oncogene. Moon,P.G.,Lee,J.E.,You,S.,Kim,T.K.,Cho,J.H.,Kim,I.S.,Kwon,T.H.,Kim,C.D.,Park, S.H.,Hwang, D.,Kim,Y.L.,Baek,M.C.,2011.Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy.Proteomics11,2459–2475.Schwenk,F.,Baron,U.,Rajewsky,K.,1995.A cre-transgenic mouse strain for the ubiquitous deletion of loxP-ﬂanked gene segments including deletion in germ cells.Nucleic Acids Res.23,5080–5081.Ypsilanti,A.R.,Zagar,Y.,Chedotal,A.,2010.Moving away from the midline:new developments for Slit and Robo.Development137,1939–1952.A.M.Krautzberger et al./Gene Expression Patterns12(2012)167–171171。

《英语感叹句》课件

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Tone: The tone of an exception sentence is usually strong and emotional, expressing surprise, exception, angle, or other intensive feelings The use of exception marks at the end of the presence reviews the emotional nature of the communication
Summary: This section teachers how to convert regular senses into exception senses and vice verse
Detail: Converting a regular presence into an exception presence manually involving adding an exception mark() at the end of the presence and using appropriate introduction to express strong emotions or emphasis a point Conversely, converting an exception sentence into a regular sentence, currently involving removing the exception mark (!) and using appropriate introduction to acknowledge the intended meaning
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VEGF受体3在乳腺癌组织中的表达及其意义_吴建忠

VEGF受体3在乳腺癌组织中的表达及其意义吴建忠1陈琳1周文斌1刘晓安1陈欣1苗登顺2【摘要】目的探讨VEGF受体3（VEGF receptor 3，VEGFR-3）在乳腺癌组织中的表达，分析其表达与乳腺癌临床病理因素的关系，为乳腺癌的临床治疗及预后判断提供依据。

方法取2004年3月－2007月6月接受乳腺癌切除术的173例女性患者乳腺癌组织存档石蜡标本（实验组），应用组织芯片技术构建乳腺癌组织芯片，HE染色判断组织芯片质量，免疫组织化学染色观察VEGFR-3表达情况；并以同期接受乳腺良性病变切除术的19例女性患者乳腺组织存档石蜡标本作为对照（对照组）。

将实验组患者按照年龄、肿瘤直径、腋窝淋巴结转移情况、病理分期以及雌激素受体、孕激素受体和人EGF受体2（human EGF receptor 2，HER-2）基因表达情况分组，比较VEGFR-3阳性表达率。

结果HE染色示两组组织芯片质量较好，能代表相应疾病组织病理学形态。

免疫组织化学染色观察示，实验组中96例（55.5%）可见VEGFR-3表达阳性，对照组均未见阳性染色。

不同年龄及雌激素受体、孕激素受体表达与否，VEGFR-3阳性表达率比较差异均无统计学意义（P > 0.05），肿瘤越大、病理分期越高，VEGFR-3阳性表达率显著提高（P < 0.05）；腋窝淋巴结转移者及HER-2基因表达阳性者VEGFR-3阳性表达率明显高于各自阴性表达者（P < 0.05）。

结论VEGFR-3在乳腺癌组织中的表达与淋巴结转移关系密切，有望成为新的判断预后指标及治疗靶点。

【关键词】乳腺癌 VEGF受体3淋巴结转移EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 3 IN HUMAN BREAST CANCER AND ITS SIGNIFICANCE/WU Jianzhong1, CHEN Lin1, ZHOU Wenbin1, LIU Xiaoan1, CHEN Xin1, MIAO Dengshun2. 1Department of General Surgery, the First Affi liated Hospital to Nanjing Medical University, Nanjing Jiangsu, 210029, P.R.China；2College of Basic Medicine, Nanjing Medical University. Corresponding author: CHEN Lin, E-mail: hxchenlin@ 【Abstract】Objective To investigate the expression and prognostic value of vascular endothelial growth factor receptor 3 (VEGFR-3) in human breast cancer. Methods A tissue microarray containing cores was constructed from 173 female patients with primary breast cancers (experimental group) and 19 female patients with benign breast lesions (control group) between March 2004 and June 2007. HE staining was performed to determine the quality of tissue microarray and immunohistochemistry staining was used to detect VEGFR-3 expression in the tissue microarray. Statistical analysis was performed to show the association between VEGFR-3 expression and clinicopathologic parameters. Results Histological examination showed that the tissue microarray had good quality, which could represent the histological characteristics of breast cancer and benign breast lesions. VEGFR-3 was expressed in 55.5% (96/173) breast cancer patients of experimental group, while the expression was negative in lesions of control group. VEGFR-3 expression rate had no signifi cant relation with age, estrogen receptor, and progesterone receptor (P > 0.05). VEGFR-3 expression rate correlated positively with tumor size and pathological staging compared to VEGFR-3 negative tumors (P < 0.05). The positive expression rate of VEGFR-3 in breast cancer with lymph node metastasis was signifi cantly higher than that without lymph node metastasis (P < 0.05). Furthermore, VEGFR-3 positive staining correlated with human EGF receptor 2 expression (P < 0.05). Conclusion The expressions of VEGFR-3 in human breast cancer have close relations with lymph node metastasis. Thus, VEGFR-3 may be a potential prognostic indicator and a new anti-cancer target for breast cancer.【Key words】Breast cancer Vascular endothelial growth factor receptor 3 Lymph node metastasisFoundation item: National Natural Science Foundation of China (81230009)乳腺癌是临床常见的女性恶性肿瘤之一[1]，淋巴DOI：10.7507/1002-1892.20130327基金项目：国家自然科学基金重点项目（81230009）作者单位：1 南京医科大学第一附属医院普通外科（南京，210029）；2 南京医科大学基础医学院通讯作者：陈琳，副主任医师，硕士生导师，研究方向：乳腺癌的基础与临床，E-mail: hxchenlin@网络出版时间：2013-11-25 15:08:50；网络出版地址：http://www. /kcms/detail/51.1372.R.20131125.1508.001.html 道转移是乳腺癌主要转移途径，腋窝淋巴结状况是决定乳腺癌分期和选择治疗方案的重要因素，也是评价预后的关键指标。

右美托咪定复合异丙酚对中重度烧伤患者围术期麻醉效果及应激因子

【摘要】目的探讨右美托咪定复合异丙酚对中重度烧伤患者围术期麻醉效果及应激因子水平的影响。

方法选择2014年9月至2016年9月本院救治的52例中重度烧伤患者为研究对象。

采用随机数表法将其分为研究组（右美托咪定复合异丙酚诱导麻醉）和对照组（异丙酚诱导麻醉），每组各26例。

比较两组患者的麻醉效果、不良反应发生率、不同时间点应激因子水平、生命体征指标变化等。

结果两组患者手术时间、术中出血量、苏醒时间比较均无显著差异（P ＞0.05），研究组患者麻醉总有效率显著高于对照组（P ＜0.05）。

研究组心率和脉搏血氧饱和度在气管插管即刻（T 1）、麻醉诱导完成后30分钟（T 2）、手术结束后（T 3）与同时间点对照组比较均无显著差异（P ＞0.05）。

两组患者T 1、T 2及T 3时间点平均动脉压均低于本组T 0时间点（P ＜0.05），研究组患者T 1、T 2及T 3时间点平均动脉压均显著高于对照组（P ＜0.05）。

两组患者术后不良反应发生率和麻醉医师满意度比较均无显著差异（P ＞0.05）。

T 3时间点，两组患者血管紧张素Ⅱ、醛固酮、皮质醇、肾上腺素水平较T 0时间点均明显升高（P ＜0.05），且研究组患者上述指标水平均明显低于对照组（P ＜0.05）。

结论右美托咪定复合异丙酚诱导麻醉可以显著提高中重度烧伤患者的麻醉效果，减轻应激反应，减少不良反应，麻醉医师满意度高，具有较好的安全性和有效性。

【关键词】右美托咪定；异丙酚；烧伤；麻醉效果；应激因子Effects of Dexmedetomidine combined with Propofol on perioperative anesthesia and expression of stress factors in moderate to severe burn patientsHUAI Qiao, JIANG Chen-xia, SUN Rui-peng, GUO Hui-yi, YANG Yan-chao, MENG Xiang-guo (Department of Anesthesiology, the First Hospital of Shijiazhuang, Shijiazhuang 050011, China)Corresponding author: MENG Xiang-guo, E-mail: mxg2008110@ 【Abstract 】 Objective To investigate the effect of Dexmedetomidine combined with Propofol on the anesthetic e ﬀect and the expression of stress factors in patients with moderate to severe burn. Method 52 cases of moderate to severe burn patients treated in our hospital from September 2014 to September 2016 were selected as subjects, and randomly divided into study group (Dexmedetomidine combined with Propofol for induction of anesthesia) and control group (Propofol induced anesthesia alone) by the random number table method. The anesthesia e ﬀects, adverse reactions incidence rates and the expression of related stress factors between the two groups were compared. Result There were no signi ﬁcant di ﬀerences in operation time, blood loss and recovery time between the two groups (P ＞0.05). The anesthetic e ﬀect of study group was signi ﬁcantly better than that of control group (P ＜0.05). At the moment of trachea cannula (T 1), 30 minutes after anesthesia induction (T 2), after the operation (T 3), there was no signi ﬁcant di ﬀerences in heart rate and SpO 2 between the two groups (P ＞0.05). MAP at T 1, T 2 and T 3 were signi ﬁcantly lower than the level at T 0 in both group (P ＜0.05), MAP in study group was signi ﬁcantly higher than that of control group at T 1, T 2 and T 3 (P ＜0.05). There were no signi ﬁcant di ﬀerences in the incidence rate of adverse reactions and the anesthesiologist satisfaction between the two groups (P ＞0.05). The vascular angiotensin Ⅱ, aldosterone, cortisol and epinephrine levels at T 3 significantly increased than the levels at T 0 in both groups (P ＜0.05), the vascular angiotensin Ⅱ, aldosterone, cortisol and epinephrine levels in study group were signi ﬁcantly lower than those in control group at T 3 (P ＜0.05). Conclusion Dexmedetomidine combined with Propofol for anesthesia in patients with moderate to severe burns can significantly improve the anesthetic effect, reduce the degree of damage on the body stress response, have less adverse reactions, th e anesthesiologists are satis ﬁed, and it has better safety and e ﬀectiveness.【Key words 】 Dexmedetomidine; Propofol; Burn; Anesthetic results; Stress factor右美托咪定复合异丙酚对中重度烧伤患者围术期麻醉效果及应激因子水平的影响怀乔，蒋晨霞，孙瑞朋，郭会艺，杨艳超，孟香果（石家庄市第一医院　麻醉科，石家庄　050011）通讯作者：孟香果　E-mail ：mxg2008110@DOI ：10.12037/YXQY .2017.05-33中重度烧伤对于机体是一种较为强烈的刺激，可引起应激反应[1]，导致机体出现一系列神经系统和内分泌系统的变化[2,3]。

PI3KAKTmTOR信号通路在糖皮质激素性股骨头坏死中的表达与作用

DOI：10.3969/j.issn.1674-2591.2021.01.014•综述・PI3K/AKT/mTOR信号通路在糖皮质激素性股骨头坏死中的表达与作用刘孟初，曹林忠，蒋玮，张琪，王多贤，马学强［摘要］糖皮质激素性股骨头坏死(steroid-induced avasculamecrosis of femoral head,SANFH)是一类致残率较高的疾病，长期损害人类与社会健康，已成为亟待解决的社会问题。

PI3K/AKT/mTOR信号通路是一条与细胞分化凋亡自噬等密切相关的转导通路。

近年随着分子生物学和细胞生物学的进步发展，表明以此信号通路为切入点能够对SANFH进行有效的靶向调控，并通过促进成骨分化，抑制凋亡，修复血管内皮细胞以及调控自噬等多种途径，对骨细胞产生显著调控和影响。

本文就PI3K/AKT/mTOR通路在SANFH中所发挥的调控机制和作用，以及各部分在激素性股骨头坏死中的表达作简要综述，为以后的研究治疗提供思路与依据。

［关键词］PI3K/AKT/mTOR；股骨头坏死；成骨细胞；破骨细胞中图分类号：R681文献标志码：AExpression and roles of PI3K/AKT/mTOR signaling pathwayin steroid-induced avascular necrosis of femoral headLIU Meng-chu,CAO Lin-zhong,JIANG Wei,ZHANG Qi,WANG Duo-xian,MA Xue-qiang Gansu University of TCM,Clinical College of Traditional Chinese Medicine,Lanzhou730030,China[Abstract]Femoral head necrosis is a kind of disease with a high risk of disability,which damages human and social health for a long time.PI3K/AKT/mTOR signaling pathway is a transduction pathway closely related to autophagy of cell differentiation and apoptosis.In recent years，with the progress of molecular biology and cell biology，it has been shown that this signal pathway can effectively target and regulate femoral head necrosis，and significantly regulate and influence osteocytes through promoting osteogenic differentiation，inhibiting apoptosis，repairing vascular endothelial cells and regulating autophagy.In this paper,the regulatory mechanism and role of PI3K/AKT/mTOR pathway in femoral head necrosis and the expression of each part in femoral head necrosis are briefly reviewed，so as to provide ideas and basis for future research and treatment.[Key words]PI3K/AKT/mTOR；necrosis of femoral head；osteoblast；osteoclast股骨头坏死是常见的一种多因素进行性病变，多是由于乙醇、类固醇激素的滥用、外伤等病因引起的局部骨组织缺血性坏死，进而导致严重的继发性髋关节病变，好发于中年人群［1］o坏死的发病机制与局部血液循环障碍、凝血障碍和骨组织再生障碍有关。

细胞氧感知机制中的低氧诱导因子(HIF-1)的生理及病理作用概述

•6•生物学教学2021年(第46卷)第3期细胞氧感知机制中的低氧诱导因子(HIF1)的生理及病理作用概述颜婷黄键*(福建师范大学生命科学学院福州350117)摘要氧是地球上绝大多数生物生命活动不可缺少的物质。

威廉•凯林、彼得•拉特克利夫和格雷格-塞门扎3位科学家因在细胞感知和适应氧含量变化机制方面做出的卓越贡献,获得了2019年诺贝尔生理学或医学奖。

本文对细胞感知和适应氧气含量变化的机制及其关键因子——低氧诱导因子1(HIR-1)在生理、病理方面的作用进行概述%关键词氧感知机制低氧诱导因子1生理病理机体生命活动需要能量，这些能量由腺昔三磷酸(ATP)提供。

人体中的ATP可以通过糖酵解和氧化磷酸化两种途径产生。

糖酵解是在无氧条件下，葡萄糖分解产生能量的方式,其产生ATP的量很少；而在有氧条件下,机体通过氧化磷酸化途径能够产生大量ATP供生命活动之需。

因此，氧对地球上绝大多数生物的生命活动不可或缺。

上世纪90年代开始，美国医学家威廉•凯林(William G.Kaelin Ja)、英国医学家彼得•拉特克利夫(Sir.Peter a.Ratcliffe)和美国医学家格雷格•塞门扎(Gregg L.Semenzz)3位科学家在细胞感知和适应氧含量变化的机制方面做出了卓越的贡献，获得了2019年诺贝尔生理学或医学奖。

本文对细胞感知和适应氧气变化的机制及其关键因子一低氧诱导因子1(hypoxia-inducibie facto-，HIR-1)在生理和病理方面的作用进行概述％1氧感知机制及其关键因子HIF-1氧感知机制揭示了在不同的含氧环境下,人体为适应环境所做出的一系列调节％该机制围绕着一个关键因子——HIF-1展开。

HIR-1是由HIR-1%和HIR-1P构成的异二聚体。

HIF-1P属组成型表达的亚基，在细胞内稳定存在，而HIR-1%则以氧依赖的方式表达。

在常氧条件下，hif-1%在脯氨酰n化酶的作用下发生n化作用，并被抑癌因子v(H)L识别和结合,结合了v(H)L的HIR-1%随即被泛素化而降解％在这个过程中，氧气的浓度可以限制n基化反应的速率，从而调节HIF-1%的积累：在缺氧条件下,HIR-1%不被降解,积累的HIR-1%进入核内，与HIF-1P结合形成二聚体后与靶基因上的缺氧反应元件(hypoxia response element,HRE)结合,在组蛋白乙酰转移酶p300等转录共激活蛋白的参与下,调节低氧条件下的相关基因表达，最终实现细胞对低氧条件的适应［1］%2HIF-1调节的生理和病理反应细胞的氧感知通路与多种生理或病理过程密切相关,特别是H1F-1作为转录因子，可调控多种基因的表达，调节相应的生理或病理反应％2.1介导糖酵解相关酶的产生，加强非氧ATP的生成氧是线粒体呼吸电子传递链的电子受体,在氧供应充足的条件下，机体主要通过有氧氧化供能％氧供应不足时,通过糖酵解可提供一部分急需能量,HIR-1是介导低氧条件下能量产生通路转换的重要因子％丙酮酸脱氢酶激酶(PDHK)、葡萄糖转运体(Glut-1)、及乳酸脱氢酶A(LDHA)等多种参与糖酵解的酶都已被证明是HIF-1介导产生的下游因子％其中，PDHK可通过磷酸化丙酮酸脱氢酶来抑制丙酮酸转化为乙酰辅酶A,从而阻断氧化磷酸化过程,抑制氧的消耗;Glut-1能加强细胞中葡萄糖的转运，为糖酵解提供底物;糖酵解相关酶的表达，既可以保证低氧条件下的能量消耗，同时又可以保护细胞不过度分解代谢％例如，早期胚胎在缺氧环境下的HIF-1可以介导激活与糖酵解有关的靶基因和与葡萄糖载体有关的靶基因，如醛缩酶A和C基因、磷酸丙糖异构酶基因、GLUT］、GLUT3等,使组织摄取和利用葡萄糖的能力增强,保证缺氧环境下胚胎细胞的能量供应［2］%肿瘤细胞如肝癌、乳腺癌等细胞的快速增长需要持续的氧气和营养供应，HIF-1通过调节GLUT］、GLUT3以及糖酵解酶类基因等的表达，提高细胞的糖酵解水平，使肿瘤细胞利用葡萄糖的能力增强，为肿瘤在缺氧条件下提供足够的营养［3］%2.2介导促红细胞生成素(EPO)基因表达，提高血液载氧能力EPO是红细胞生成的主要调节物，主要由肾脏和肝脏，特别是肾脏产生％HIR-1介导了EPO的氧依赖性调控％缺氧、贫血、肾血流减少等任何引起肾氧供应不足的因素,都会引起肾皮质肾小管周围的间质细胞产生HIF-1,HIR-1与EP0基因3'端缺氧应答原件的增强子结合，促进EPO表达［4］%EPO与骨髓造血细胞上的相应受体结合，通过促进红系祖细胞的有丝分裂、激活血红蛋白特异基因的表达、抑制晚期红系祖细胞的凋亡以及促进网织红细胞的增殖和释放等机制，诱导红细胞大量增殖,增加机体的携氧能力，从生物学教学2021年（第46卷）第3期-7-而缓解缺氧带来的一系列不良反应。

HIF-1α和TGF-β1在乳腺癌中作用的研究进展

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重要的参考价值，有望在未来成为乳腺癌诊断及评估的一个重要指标。
关键词：ＨＩＦ－１α；ＴＧＦ－β１；ＥＭＴ；乳腺癌
上皮间质转化在多种癌前病变中存在，可以通过影响上
皮细胞之间的黏附能力，导致其黏附能力下降，进而对于细
胞运动与迁移能力产生影响，同时也会使细胞形态在一定程
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重要恶性肿瘤之一。目前，乳腺癌的病因与发病机制并未十分明确。大多研究报告，生物机体内肿瘤的形成及恶化转移与人体

康柏西普联合手术治疗新生血管性青光眼的疗效评价

• 82 •实用临床医药杂志Journal of Clinical Medicine in Practice2018年第22卷第9期康柏西普联合手术治疗新生血管性青光眼的疗效评价杨文，谭英，卢国华，顾培(南京医科大学附属常州第二人民医院，江苏常州，213164)摘要：目的探讨康柏西普联合手术治疗新生血管性青光眼的临床疗效。

方法选取新生血管性青光眼患者10例(0眼），随机分为2组，实验组予以康柏西普玻璃体腔内注射联合小切除以及全视网膜激光光凝术（P R P)治疗，对照组采用阿托品等药物联合外科手术常规治疗。

比较2组疗效、眼压变化、视力改善、房水中血管内皮生长因子（V E G F)表达水平。

结果2组患者治疗后视力均明显提升。

2组患者治疗前后眼压显著明显（P<0.05)。

2组血清以及房水中V E G F表达水平均显著降低，且实验组V E G F下降水平显著更低(P<0.05)。

结论康柏西普结合外科手术可以有效改善新生血管性青光眼患者临床症状，降低眼压。

关键词：康柏西普；新生血管性青光眼；玻璃体；全视网膜激光光凝术；眼压；血管内皮生长因子中图分类号：R775 文献标志码：A文章编号：1672-2353(2018)09-082-04 D O I: 10.7619/j c m p.201809022Mfect of conbercept combined witli operation ontreatment of patients with neovascular glaucomaY A N G W e n，T A N Y in g，LU G u o h u a，G U Pei(Changzhou Second People's Hospital Affiliated to Nanjing Medical University，Changzhou，Jiangsu，213164)A B S T R A C T：O bjective To explore the effect of conbercept com bined with operation on treatm ent of patients with n eovascular glaucom a. M ethods A total of 10 patients with neovascular glaucoma (10 eyes)were selected and randomly divided i nto experim ental group and contro experim ental group was treated with intravitreal injection of conbercept plus trabeculectom y and panretinal photocoagulation (P R P), while the control group was treated with atropine, glucocorticoid and panretinal photocoatulation. The pre-treatm ent and post-treatm ent visual acuity，intraocular pressureand vascular endothelial g rowth factor ( V E G F)expression in serum and hum or aqueous were compared. Results After treatm ent，the visual acuity improved significantly (P <0. 05). The intraocular pressure of the experim ental group and control group before treatm ent and after treatm ent decreased significantly (P<0. 05). The expression level of the post-treatm ent VEGF in serum and hum or aqueous in the experim ental group and control group decreased significantly, and im ental group was m uch more lower ( P< 0. 05 ). Conclusion Application of conbe with surgery can effectively improve the clinical symptoms and reduce intrao with neovascular glaucoma.K E Y W O R D S：conbercept ；neovascular glaucom a;vitreous body;panretinal photocoatulation ；intraocular pressure;vascular endothelial growth factor血管内皮生长因子（V E G F)是一种与肿瘤生长以及转移关系密切的糖蛋白化合物，研究[14发现，V E G F可以通过与血管内皮细胞生长因子受体结合，促进内皮细胞的有丝分裂，并导致内皮细胞大量的新生，发挥促进新生血管形成的作用。

血浆同型半胱氨酸和C反应蛋白联合检测在颈动脉粥样硬化诊断中的应用

血浆同型半胱氨酸和C反应蛋白联合检测在颈动脉粥样硬化诊断中的应用目的探討血浆同型半胱氨酸（Hcy）和C反应蛋白（CRP）检查对诊断颈动脉粥样硬化的意义和效果。

方法选取2015年1月～2017年1月来我院就诊的70例颈动脉粥样硬化确诊患者作为观察组，同时进行血管外超声，将颈动脉内膜中层厚度局限性增厚但无斑块形成的40例患者设为A组（无斑块形成），将颈动脉内膜中层厚度弥漫性增厚且可见斑块形成的30例患者设为B组。

随机选择来我院体检中心体检未发现颈动脉粥样硬化的70例患者做为对照组。

检测并且比较观察组和对照组患者的Hcy、CRP及其他生化指标水平。

结果观察组和对照组患者的血清尿素氮、肌酐、尿酸、空腹血糖、三酰甘油、肌酸激酶、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇的水平比较，差异无统计学意义（P>0.05）。

观察组患者的Hcy和CRP水平显著高于对照组，差异有统计学意义（P＜0.05）。

A组的Hcy与CRP水平显著低于B组，差异有统计学意义（P＜0.05）。

结论 Hcy和C反应蛋白联合检查可以为诊断劲动脉粥样硬化提供诊断依据，是一种快速有效的检测指标，临床上可普及该项检查。

To investigate the significance and effect o f plasma [Abstract]Objective（CRP） in the diagnosis of carotid homocysteine （Hcy） and C-reactive proteindiagnosed i n our70 patients with carotid atherosclerosisatherosclerosis.Methods，hospital from January 2015 to January 2017 were selected as the observation group，40 patients with carotidat the same time，extravascular ultrasound was performedintima-media thickness of the localized thickening but no plaque formation were），30 patients with the carotid artery intima（no plaque formationselected as group Athick d iffuse thick a nd visible plaque f ormation was selected as group B.70 casesin our hospital physical examination c enter w erewithout c arotid atherosclerosisrandomly selected as the control g roup.The levels o f Hcy，CRP，and otherbiochemical markers in the observation group and the control groups were measuredand compared.Results There was no significant difference in the levels of serum urea，creatine kinase，，triglyceride，fasting blood glucosenitrogen，creatinine，uric acidbetween the observation grouphigh density lipoproteinand low density lipoprotein（P>0.05）.The levels of Hcy and CRP in the observation groupand the control group，with significant differencewere significantly higher than those in the control grouplower than（P＜0.05）.The levels of Hcy and CRP in group A were significantly（P＜0.05）.Conclusionthose in group B，the difference was statistically significantPlasma Hcy and C-reactive protein can provide a diagnostic basis for the diagnosis ois a quick and effective method，which is suggested t hat t he atherosclerosis.Itexamination can be popular used clinically.；；Carotid atherosclerosis；C-reactive protein[Key words]Plasma homocysteineDiagnosis；Significance颈动脉硬化严重时常伴有有硬化斑块脱落[1]，甚至会阻塞大脑血管，临床上常用影像学方法进行检查确诊，如颈动脉超声波检查[2]、磁共振颈动脉成像[3]、颈动脉血管造影等[4]。

法舒地尔对血管内皮损伤保护机制的研究及安全性分析

·综述·法舒地尔对血管内皮损伤保护机制的研究及安全性分析李颖1张和平1曾小凤1汪姝婷1唐金城1冯江超1【摘要】动静脉内瘘功能不良与血管内皮损伤程度密切相关。

动静脉内瘘手术中的损伤及术后动静脉内血流力学改变均可造成血管内皮不同程度的损伤，保护损伤的血管内皮细胞是防治早期内瘘功能不良的关键。

法舒地尔(Fasudil)是一种Rho激酶抑制剂，能降低炎症反应、缓解血管痉挛、内皮功能紊乱、预防血栓形成、扩张血管等，进而保护血管内皮损伤，但其机制并不完全清楚。

本文综述了法舒地尔血管保护机制，为动静脉内瘘术后法舒地尔临床应用提供依据。

【关键词】法舒地尔；血管内皮损伤；安全性；动静脉内瘘术中图分类号：R459.5文献标识码：A doi:10.3969/j.issn.1671-4091.2021.05.010The mechanism and safety of Fasudil on the protection of vascular endothelia from injury LI Ying1,ZHANG HE-ping1,ZENG Xiao-feng1,WANG Shu-ting1,TANG Jin-cheng1,FENG Jiang-chao1Department ofNephrology,The Affiliated Hospital of North Sichuan Medical College,Sichuan637000,ChinaCorresponding author:FENG Jiang-chao,Email:*****************【Abstract】Reginal vascular endothelial injury is closely related to the dysfunction of arteriovenous fistu-la.Surgical injury and hemodynamics changes after the surgery may cause vascular endothelial injury of vari-ous degrees.Therefore,to protect vascular endothelia from injuries is the key to preserve early function of thearteriovenous fistula.Fasudil,a Rho kinase inhibitor,relieves inflammatory reactions,vasospasm,endothelialdysfunction,thrombosis,vasodilatation,and many other functions to protect vascular endothelial cells from in-juries.However,the mechanism of Fasudil remains controversial.This article reviews the vascular protectionmechanism of Fasudil and provides the basis for clinical application of Fasudil after arteriovenous fistula sur-gery.【Key words】Fasudil;Vascular endothelial injury;Safety;Autologous arteriovenous fistula自体动静脉内瘘(autologous arteriovenous fistula，AVF)是终末期肾病患者重要的血管通路，具有安全可靠、血流量充分、感染率低、维持时间长、易于反复穿刺、对患者日常生活影响小等优点[1]，但AVF术可诱发血管内皮细胞损伤，触发炎症因子激活，血管痉挛等，并且管腔内血流动力学改变，血管壁剪切力上升，进一步损伤内瘘的内皮功能。

毛刺征联合血管集束征在肺磨玻璃样结节良恶性鉴别诊断中的应用价值

2021，49（6）：718-720.[18]邢燕，刘小玲.肝硬化食管静脉曲张患者初次内镜治疗后出血发生状况及其影响因素[J].航空航天医学杂志，2021，32（9）：1075-1077.[19] MATEI D，CRISAN D，PROCOPET B，et al.Predictivefactors of failure to control bleeding and 6-week mortality aftervariceal hemorrhage in liver cirrhosis-a tertiary referral center experience[J].Arch Med Sci，2021，18（1）：52-61.[20]连佳，韩涛，向慧玲，等.肝硬化食管胃静脉曲张出血患者胃镜治疗术后再出血的影响因素分析[J].临床肝胆病杂志，2021，37（9）：2092-2096.（收稿日期：2023-02-23）　（本文编辑：何玉勤）①江西省新余市人民医院　江西　新余　338000通信作者：阮玖根毛刺征联合血管集束征在肺磨玻璃样结节良恶性鉴别诊断中的应用价值廖建①　阮玖根①　肖琼①【摘要】　目的：探究毛刺征联合血管集束征在肺磨玻璃样结节良恶性鉴别中的应用价值。

方法：选取新余市人民医院2019年4月—2022年4月收治的113例肺磨玻璃样结节患者，入院后均进行多层螺旋CT（MSCT）检查，按照穿刺或术后病理活检结果将其分为良性组（n =45）和恶性组（n =68）。

比较两组患者一般临床资料及MSCT 影像学特征，以穿刺或术后病理活检结果为金标准，比较毛刺征、血管集束征单独及联合诊断恶性肺磨玻璃样结节的准确度、敏感度、特异度。

结果：两组患者的性别、年龄及吸烟史比较，差异均无统计学意义（P >0.05）；恶性组肿瘤家族史占比高于良性组，差异有统计学意义（P <0.05）。

与良性组相比，恶性组结节高密度、形状不规则、边界粗糙、毛刺征、血管集束征、胸膜凹陷征占比均更高，病灶直径更长，差异均有统计学意义（P <0.05）。