IgA肾病小鼠模型的建立与鉴定
KiM-1小鼠模型的构建及其肾纤维化机理研究的开题报告
KiM-1小鼠模型的构建及其肾纤维化机理研究的开题报告1. 研究背景和意义肾纤维化是一种常见的肾脏病变，常见于肾小球肾炎、糖尿病肾病等疾病的晚期。
其主要特点是肾小球滤过膜及间质区出现大量的胶原增生及基质沉积，导致肾小球滤过率下降，最终发展为肾功能衰竭。
目前尚无有效的治疗手段，因此肾纤维化的机理研究具有重要的理论和实践意义。
在肾纤维化的发病过程中，紧密连接蛋白-1（kim-1）在病理机制中起着重要的作用。
KiM-1是一种跨膜糖蛋白，主要分布于肾小管上皮细胞表面，并在肾损伤后被高表达。
近年来的研究表明，KiM-1在肾纤维化的发生和发展过程中具有重要作用。
因此，对KiM-1作为肾纤维化分子机制的研究具有重要意义。
2. 研究目的本文旨在构建KiM-1小鼠模型，通过对该模型的实验研究，分析和探究KiM-1在肾纤维化的发生和发展中所扮演的作用机制，为肾纤维化的研究提供新的思路和理论支持。
3. 研究内容和方法3.1 实验材料及设备KiM-1小鼠（C57BL/6J）及愈创木酚模型制备所需药品（篝火酸、巴曲酸、对氨基水杨酸），肾脏相应组织采集及处理所需试剂（RNAlater、甲醛、蜡块等），免疫组织化学染色试剂盒（DAB、抗KiM-1抗体等），qPCR试剂盒（SYBR Green、cDNA转录试剂盒等），Western blot试剂盒（SDS-PAGE凝胶、蛋白质电泳装置等），显微镜、腹腔穿刺针、注射器等。
3.2 实验步骤（1）KiM-1小鼠模型的构建：通过腹腔注射愈创木酚，制备KiM-1小鼠模型。
（2）组织采集及处理：在实验结束后，分别采集小鼠的肾组织，并用RNAlater、甲醛、蜡块等试剂进行处理。
（3）免疫组织化学染色：制备肾脏组织切片，使用抗KiM-1抗体进行免疫组织化学染色，并通过显微镜观察染色结果。
（4）基因表达分析：通过qPCR方法对KiM-1等基因进行表达分析，探究其在肾纤维化中所扮演的作用。
（5）Western blot分析：通过Western blot检测KiM-1在肾脏组织中的表达水平，进一步验证其在肾纤维化中的作用。
纳豆多糖改善小鼠IgA肾病的作用及其免疫学机制的研究分析
纳豆多糖改善小鼠IgA肾病的作用及其免疫学机制的研究分析佟芳;毛成健【摘要】目的探讨纳豆多糖改善小鼠IgA肾病的作用及其免疫学机制.方法将造模成功的50只IgA肾病小鼠分为模型组,纳豆多糖高、中、低剂量组及阳性组(雷公藤多苷),每组10只.同时选择10只正常小鼠(未经造模)作为对照组.正常组和模型组小鼠按0.2 mL/10g 灌胃生理盐水(NS),阳性组(雷公藤多苷,5mg/kg),纳豆多糖高、中、低剂量组(给药剂量分别为40 、20、10 g/kg),1次/d,至第20周处死,取血及脏器,测定白蛋白、尿素氮、肌酐、血清循环免疫复合物和IgA水平.结果与正常组比较,模型组尿微量白蛋白明显升高(P<0.05);与模型组比较,纳豆多糖高、中剂量组和阳性组尿微量白蛋白明显降低(P<0.05);与正常组比较,模型组尿血清白蛋白明显降低(P<0.05),与模型组比较,纳豆多糖高、中剂量组和阳性组血清白蛋白明显升高(P<0.05);与正常组比较,模型组尿素氮、肌酐、血清循环免疫复合物明显升高(P<0.05),与模型组比较,纳豆多糖高、中剂量组和阳性组尿素氮、肌酐、血清循环免疫复合物明显降低(P<0.05);与正常组比较,模型组肾组织IgA水平明显升高(P<0.05),与模型组比较,纳豆多糖高、中剂量组和阳性组IgA水平明显降低(P<0.05).结论纳豆多糖可明显改善小鼠IgA肾病,还可降低尿蛋白和IgA水平.%Objective To observe the protective effect of natto polysaccharide on IgA nephropathy mice and its immunological mechanism.Methods The IgA model mice were divided into model group, high-, medium-and low-doses of natto polysaccharide groups, and positive group.Each group had 10 mice.10 normal mice were enrolled as normal control group.The mice in normal control and model groups were treated with normal saline, and alldoses of natto polysaccharide groups and positive group were treated with natto polysaccharide (40, 20, 10 g/kg) and positive (5mg/kg) respectively.All mice were treated with drugs or NS for 20 weeks, ig,qd.The levels of albumin in serum and urine, urea nitrogen, creatinine, CIC and IgA were investigated in each group.Results Compared with the normal control group, the levels of albumin in urine in model group were obviously higher (P<0.05).but compared with the model group, the levels of albumin in urine in high-, medium-doses of natto polysaccharide groups and positive group were obviously lower (P<0.05).Compared with the normal control group, the levels of albumin in serum in model group were obviously lower (P<0.05), but compared with the model group, the levels of albumin in serum in high-, medium-doses of natto polysaccharide groups and positive group were obviously higher (P<0.05).Compared with the normal control group, the levels of urea nitrogen, creatinine and CIC in model group were obviously higher (P<0.05), but compared with the model group, the levels of urea nitrogen, creatinine and CIC in high-, medium-doses of natto polysaccharide groups and positive group were obviously lower (P<0.05).Compared with the normal control group, the levels of IgA in model group were obviously higher (P<0.05), but compared with the model group, the levels of IgA in high-, medium-doses of natto polysaccharide groups and positive group were obviously lower (P<0.05).Conclusion Natto polysaccharide has protective effect on IgA nephropathy mice, and can reduce the levels of albumin and IgA.【期刊名称】《标记免疫分析与临床》【年(卷),期】2017(024)006【总页数】4页(P706-708,712)【关键词】纳豆多糖;IgA肾病;免疫学【作者】佟芳;毛成健【作者单位】湖北省宜昌市第二人民医院三峡大学第二人民医院肾内科,湖北宜昌443000;湖北省宜昌市第二人民医院三峡大学第二人民医院肾内科,湖北宜昌443000【正文语种】中文IgA肾病是一种与细胞免疫调节紊乱有关的肾小球系膜区出现IgA免疫复合物沉积的原发性肾小球肾炎。
IgA肾病在老鼠的动物模型（学习资料）
JN EPHROL 2008; 21: 463-467THOROUGH CRITICAL APPRAISAL 463A BSTRACT IgA nephropathy is the most common primary chronic glomerulonephritis, and was first described by J. Berger (Transplant Proc. 1969;1:939-944). Histopathologically, IgAnephropathy is characterized by expansion of glomerular mesangial matrix, with mesangial cell proliferation.Glomeruli typically contain generalized-diffuse granular mesangial deposits of IgA, IgG and C3. Since pathogene-sis of IgA nephropathy is still obscure, it is important to tryto determine the initiation and progression of this disease using a suitable animal model. Several investigators, in-cluding Rifai’s group (Rhode Island, USA) and Emancipa-tor’s group (Cleveland, Ohio, USA), reported various exper-imental animal models for this disease. In 1985, Imai et al first reported that the ddY strain of mouse can serve as a spontaneous animal model for IgA nephropathy. These mice show mild proteinuria without hematuria, and mesangioproliferative glomerulonephritis with severe glomerular IgA deposits in association with an increase of serum IgA level (Imai et al. Kidney Int. 1985;27:756-761).Electron-dense deposits are observed in the glomerular mesangial areas by electron microscopy. Furthermore,Muso’s group succeeded in generating a mouse model of IgA nephropathy with a high incidence and early onset of glomerular IgA deposition (Miyawaki et al. Nephron.1997;76:201-207). The selection procedure was successful in increasing the serum IgA level of the selected line. The selected ddY line (HIGA mice) showed only mild protein-uria (100-300 mg/dL ) and did not show hematuria. These immunohistopathological findings in ddY mice resemble those in IgA nephropathy patients. The objectives of this review are to introduce the genetic background, Th1/Th2polarization, expansion of extracellular matrices (ECMs)and treatment of IgA nephropathy of the ddY mouse.These findings from the ddY mouse appear to be useful indetermining the pathogenesis and treatment of patients with IgA nephropathy.Key words:ddY mouse, IgA nephropathy, Pathogenesis,Treatment H ETEROGENEITY OF GENETIC BACKGROUND AND T H 1/T H 2 POLARIZATION IN THE DD Y MOUSEGenetic backgroundDeposition of the major retroviral envelop glycoprotein, gp 70,in glomeruli of ddY mice was examined by immunofluores-cence. Takeuchi et al (1) reported that gp70 was deposited inthe glomerular mesangial areas in ddY mice over 24 weeks,in the same way as IgG and IgA deposits. However, positive staining of gp70 was not observed in glomeruli of our strain of ddY mice at any age, whereas deposition of IgA, IgG and IgM was marked in mice aged over 40 weeks. It appears that gp70 deposition may not be the sine qua non for the patho-genesis of IgA nephropathy in ddY mice (2).Genetic factors are considered to be involved in the patho-genesis of IgA nephropathy on the basis of racial differences in prevalence and familial aggregation. Serial renal biopsies were performed at 20, 40 and 60 weeks of age in 361 ddY mice. These mice were classified into 3groups on the basisof onset of glomerular injury as follows: early onset at 20weeks (31.9%), late onset at 40 weeks (37.9%) and quies-cence at 60 weeks (30.2%). The severity of glomerular injuries in both onset groups correlated with the intensity of glomerular I gA deposition but not with serum I gA levels. A genome-wide scan using 270 microsatellite markers identi-fied 3chromosomal regions on chromosomes 1, 9 and 10,which were significantly associated with the glomerular injuries. Surprisingly, the peak marker D10MIT86 on chromo-some 10 is located on the region synthenic to 6q22-23 with IGAN1, which might be responsible for familial IgA nephropa-thy (3, 4). In addition,D1MIT16 on chromosome 1 was locat-ed very close to the locus of the selectin gene, which is aknown candidate for human IgA nephropathy. It appears thatthe 3-group ddY mouse model can be a useful tool for identi-fying susceptibility genes and also for examining their roles inthe pathogenesis of IgA nephropathy.Yasuhiko Tomino Division of Nephrology, Department of Internal Medicine,Juntendo University School of Medicine, Tokyo - JapanIgA nephropathy: lessons from an animal model,the ddY mouse/jnonline –Th1/Th2 polarizationSeveral studies noted that functional abnormalities of T and/or B cells may be involved in the pathogenesis of IgA nephropathy. Th1/Th2 imbalance is expected to play an important role in this disease. I masawa et al (5) of the Sakai group and I wata et al (6) demonstrated that in a patient with I gA nephropathy and chronic myeloblastic leukemia, bone marrow transplantation (BMT) resulted in remission of not only leukemia but also of IgA nephropa-thy.I masawa et al also reported that BMT from normal mice attenuated glomerular lesions in high–serum I gA (HI GA) ddY mice, although glomerular lesions with I gA deposits were reconstituted in normal recipient mice after BMT from HIGA mice (7). Suzuki et al (8)(H. Suzuki is one of my colleagues)transplanted bone marrow cells (BMCs) from 20-week-old ddY mice with early or quiescent I gA nephropathy into similar irradiated ddY mice, C57Bl/6 (Th1 prone) mice or BALB/c (Th2 prone) mice. Serum IgA/IgG complex and Th1/Th2 polarization of spleen cells was determined by enzyme-linked immunosorbent assay and confirmed by fluorescent cytometric analysis. As a result, the ddY mice with early I gA nephropathy demon-strated strong polarization toward Th1, while those with quiescent disease were Th2 polarized. Serum levels of I gA/I gG2a immune complex significantly correlated with the severity of the glomerular injuries. Bone marrow taken from mice with early I gA nephropathy caused I gA nephropathy with Th1 polarization in recipient, quiescent mice, while that in quiescent mice was polarized Th2. BMT from the quiescent ddY mice ablated glomerular injury and mesangial IgA/IgG deposition in such mice with early I gA nephropathy. I t appears that BMCs, presumed to be IgA-producing cells, may initiate this disease. Thus, Th1 cells may be involved in the pathophysiology of the disease after glomerular IgA deposition in IgA nephropa-thy patients. This study showed that the quiescent mice were Th2 prone, while the onset ddY mice had strong polarity, suggesting that both Th1 and Th2 polarities may contribute to different processes of disease, such as initi-ation and progression, respectively.Expansion of extracellular matrixThe correlations between the steady-state mRNA levels of extracellular matrix(ECM)using specific cDNA probes for the a1 (I V) chain;laminin A, B1 and B2 chains; heparan sulfate proteoglycan (HSPG);and glomerular injuries in ddY mice were evaluated. The intensity and distribution of type IV collagen, laminin and HSPG in ddY mice were examined by immunofluorescence. I ncreased expression of ECM genes by the a1 (IV) chain;laminin A, B1 and B2 chains;and HSPG was observed in renal tis-sues of ddY mice. Staining of type I V collagen, laminin and HSPG was observed in renal tissues of ddY mice. Increased proteinuria in 40-week-old ddY mice might be related to the decrease in glomerular HSPG, which acts as the anionic site in such areas. Marked proliferation and/or expansion of glomerular resident cells and mesan-gial matrices were observed in 40-week-old ddY mice. The intensity of IgA and C3 deposits in the glomeruli was in parallel with the levels of mRNA for such ECM compo-nents. I t appears that increased mRNA levels for such matrices coincided with the development of renal injuries in ddY mice (9).It is not known, however, whether IgA deposits influence the expression of ECM components in patients with I gA nephropathy. We reported that deposits of IgA and/or C3 did not influence the major components of glomeruli in patients with I gA nephropathy. I t can be concluded that the initiating factors of the collapse and/or sclerosis of glomeruli might be factors other than the glomerular deposition of I gA in patients with I gA nephropathy. We have previously evaluated whether treatment with mono-clonal antibody to murine CD4 molecules (mAb CD4) modulates the production of ECM in glomeruli of ddY mice (10). To determine the role of I gA deposits in the pathogenesis of IgA nephropathy, immunofluorescence of type IV collagen, fibronectin and HSPG was performed in renal tissues of mAb CD4–treated and saline control ddY mice. Rat IgG2 mAb CD4 (GK 1.5) was a kind gift of Dr. L.A.Herzenberg of Stanford University and was originally provided by Dr.Frank W. Fitch, University of Chicago. During the experimental periods, ddY mice were treated with mAb CD4 at 2 mg (0.2 mL). In immunofluorescence, the mean intensity of IgA deposits in glomeruli of the mAb CD4–treated ddY mice was significantly lower than that in saline-treated control mice. Glomerular mesangial expan-sion in the treated mice was milder than that found in the saline control ddY mice. However, there was no signifi-cant difference in the intensity or distribution of ECM components in glomeruli between the treated mice and the saline control mice at 8-40 weeks of age. Thus, the reduction of glomerular mesangial expansion observed in the mAb CD4–treated ddY mice might be due to the decrease of glomerular I gA deposits. I t appears that glomerular I gA deposits might not induce production of ECM components in IgA nephropathy of ddY mice.464Tomino: Spontaneous mouse model of IgA nephropathyT REATMENT OF I G A NEPHROPATHY IN THE DD Y MOUSEDiet therapyMy group attempted to compare glomerular changes between low-protein and high-protein diets in ddY mice (11). ddY mice were fed a standard diet containing 22% protein until 40 weeks of age. Marked depositions of IgA and C3 in glomeruli and glomerular expansion were observed in ddY mice after 40 weeks of age. These mice were divided into 2diet groups:i.e.,low-protein (6%) and high-protein (50%). The mice of both groups were sacri-ficed at 70 weeks of age. At each time of measurement after 50 weeks of age, the level of urinary protein excretion in low-protein diet mice was significantly decreased com-pared with that in high-protein diet mice (p<0.01). Glomerular enlargement and mesangial expansion were improved in the low-protein diet mice. I ntensities of I gA and C3 in glomeruli of the low-protein diet mice were sig-nificantly lower than those in high-protein diet mice. It was confirmed that dietary protein restriction is useful for the prevention of glomerular injuries, even when such therapy is started after the appearance of IgA nephropathy in ddY mice.Drug therapyTreatment with a monoclonal antibody to murine CD4 moleculesImmunopathological studies were performed to determine whether glomerular injuries in ddY mice are influenced by treatment with a mAb to murine CD4 molecules as described above (10). The ddY mice were initially treated with intravenous injections, followed by weekly intraperi-toneal injections of mAb CD4. Flow cytometry showed that there was a marked decrease in the number of CD4+ T cells. In immunofluorescence, the mean intensity of IgA deposits in the glomerular mesangial areas and capillary walls of treated ddY mice was significantly lower than that in saline-treated control ddY mice of comparable age. Glomerular mesangial expansion in the treated ddY mice was milder than that found in the same control ddY mice. However, no significant differences in the levels of serum I gA, urinary protein excretion and average number of intraglomerular cells were observed between the treated and control ddY mice. I t appears that although CD4+T cells control the amount of IgA deposits in glomeruli, other factors may be involved in the evolution of IgA nephropa-thy in ddY mice. I t is not known whether the increase observed in the number of intraglomerular cells in both the treated and control ddY mice is due to resident glomerular cells or mononuclear cells infiltrating the glomeruli. Our studies showed that increased glomerular cells in ddY mice are positive for markers of Thy-1.2 (total T cells), CD8 (killer/suppressor T cells) and CD11 (mac-1, macrophages/monocytes), suggesting that the majority of these cells are likely to be infiltrating cells and glomerular mesangial cells. Because several cytokines or growth fac-tors,such as IL-1,IL-6,TNF-a and platelet-derived growth factor (PDGF),have been shown to be involved in mesan-gial cell proliferation, it was suggested that, in addition to the effect of CD4+T cells that may modulate the amount of glomerular IgA deposits, these factors may be involved in the progressive mechanism of IgA nephropathy.MizoribineMizoribine, an immunosuppressant, was developed in Japan and shown to prevent the proliferation of lympho-cytes in vitro and to possess immunosuppressive action in vivo. Since mizoribine also has a suppressive effect on antibody formation via direct inhibition of B-cell function, it has beneficial effects in patients with chronic glomeru-lonephritides, lupus nephritis, nephrotic syndrome and renal transplantation. Shimizu et al (12), my colleagues, determined the clinical and immunopathological effects of mizoribine in ddY mice. The ddY mice were treated with 0.05 mg/mL (low dose) or 0.1 mg/dL (high dose) of mizoribine for 35 weeks. Numbers of total T cells (CD3+T cells), CD4+T cells, CD8+T cells and CD11b+cells among the spleen cells were measured by flow cytometry. Numbers of I gA-, I gG- or I gM-bearing B cells among spleen cells were also calculated. Immunohistopathologi-cal changes were examined by light microscopy and immunofluorescence. After 20 weeks of treatment, levels of urinary protein excretion in the ddY mice treated with the high dose of mizoribine were lower than those treated with the low dose. Expansion of glomerular mesangial areas in ddY mice treated with the high dose of this drug was significantly decreased compared with the low dose or with the drinking-water control. Numbers of B cells and I gA-bearing B cells among the spleen cells of ddY mice treated with the low or high dose of mizoribine were lower than in those given only drinking water. I t appears that treatment with mizoribine might affect B cells, especially465JN EPHROL2008; 21: 463-467I gA-bearing B cells, and improve glomerular injury in I gA nephropathy of ddY mice.Endothelin receptor antagonists Endothelin (ET) is a possible mediator of renal disease progression. Several investigators have previously report-ed that mRNA levels of ETs are abnormally regulated in diseased kidneys. ETs contribute to the progression of glomerular disease either by inducing local vasoconstric-tion or by stimulating mesangial cell proliferation and excess synthesis of ECM components. Nakamura et al (13) examined whether a specific endothelin receptor A (ETA)antagonist (FR139317;Fujisawa Pharmaceutical,Osaka, Japan) affects the progression of glomerulonephri-tis in ddY mice. ddY mice were injected intraperitoneally with 3.0mg/100g body weight per day of FR139317 for 36 weeks. At 24 weeks of age, ddY mice were divided into 2groups that received intraperitoneally either FR139317or its vehicle (saline) daily for 36 weeks. The development of histopathological lesions and urinary protein excretion were suppressed by FR139317 treatment. It was suggest-ed that treatment with the ETA antagonist FR139317 was effective in ddY mice with IgA nephropathy.Steroid-liposomeImmunopathological studies were performed to determine whether glomerular injuries in ddY mice are influenced by treatment with a newly developed liposome loaded with prednisolone phosphate (PSL-liposome)(14). The newly synthesized novel cationic lipid 3,6-dipentadeciroxy-1-amizino-benzene (TRX-20) was employed to obtain selec-tive affinity to the anionic cell surface and ECM in glomerular mesangial lesions. ddY mice were treated intravenously with 1.0 mg/kg of PSL-liposome once a week from 45 weeks to 61 weeks of age. ddY mice werealso intravenously treated with 1.0 mg/kg of ordinary PSLonce a week. I n immunofluorescence, mean intensity ofI gA and C3 depositions in glomeruli of PSL-liposome–treated ddY mice were markedly decreased when compared with those of ordinary PSL-treated and untreated control ddY mice. Glomerular mesangial expan-sion in PSL-liposome–treated ddY mice was milder than that in ordinary PSL-treated ddY mice or untreated control ddY mice. It appears that treatment with PSL-liposome is effective in improving glomerular I gA and C3 depositionsand glomerular expansion in I gA nephropathy of ddY mice. I t is necessary to determine whether steroid-lipo-some treatment is more effective than ordinary steroids in patients with IgA nephropathy.A CKNOWLEDGEMENTS I sincerely thank my colleagues in the Division of Nephrology at Juntendo University, Tokyo, Japan. Conflict of interest statement: None declared.Address for correspondence:Yasuhiko Tomino, MD Division of Nephrology Department of Internal Medicine Juntendo University School of Medicine Hongo 2-1-1, Bunkyo-KuTokyo 813-8421, Japan*****************.ac.jp 466Tomino: Spontaneous mouse model of IgA nephropathy R EFERENCES 1. Takeuchi E, Doi T, Shimada T, Muso E, Maruyama N,Yoshida H. Retroviral gp 70 antigen in spontaneous mesan-gial glomerulonephritis of ddY mice. Kidney nt.1989;35:638-646.2. Shimizu M, Tomino Y , Abe M, Shirai T, Koide H. Retroviral envelope glycoprotein (gp 70) is not a prerequisite for pathogenesis of primary immunoglobulin A nephropathy inddY mice. Nephron. 1992;62:328-331.3. Gharavi AG, Yan Y , Scolari F , et al. I gA nephropathy, themost common cause of glomerulonephritis, is linked to6Q22-23. Nat Genet. 2000;26:354-357.4. Suzuki H, Suzuki Y , Yamanaka T, et al. Genome-wide scan in a novel IgA nephropathy model identifies a susceptibilitylocus on murine chromosome 10, in a region synthenic tohuman I GAN1 on chromosome 6Q22-23. J Am SocNephrol. 2005;16:1289-1299.5. Sakai O. I gA nephropathy: current concepts and futuretrends. Nephrology. 1997;3:2-3.6. I wata Y, Wada T, Uchiyama A, et al. Remission of I gAnephropathy after allogeneic peripheral blood stem cell transplantation followed by immunosuppression for acute lymphocytic leukemia. Intern Med. 2006;45:1291-1295.7.Imasawa T, Nagasawa R, Utsunomiya Y, et al. Bone marrowtransplantation attenuates murine IgA nephropathy: role of a stem cell disorder. Kidney Int. 1999;56:1809-1817.8. Suzuki H, Suzuki Y, Aizawa M, et al. Th1 polarization inmurine I gA nephropathy directed by bone marrow-derived cells. Kidney Int. 2007;72:287-291.9. Tomino Y, Nakamura T, Ebihara I, et al. Altered steady-statelevels of mRNA coding for extracellular matrices in renal tis-sues of ddY mice, an animal model for IgA nephropathy. J Clin Lab Anal. 1991;5:106-113.10. Tomino Y, Shimizu M, Koide H, Abe M, Shirai T. Effect ofmonoclonal antibody CD4 on glomerulonephritis of ddY mice, a spontaneous animal model of IgA nephropathy. Am J Kidney Dis. 1993;21:427-432.11. Ohmuro H, Shimizu M, Tsushima Y, et al. Effect of low-pro-tein diet on glomerular changes in ddY mice: a spontaneous animal model of I gA nephropathy. Nephron. 1996;72:333-334.12. Shimizu M, Shou I, Tsuge T, Abe M, Tomino Y. Effect ofmizoribine on glomerulonephritis of early-stage I gA nephropathy in ddY mice. Nephron. 1998;79:67-72.13. Nakamura T, Ebihara I, Fukui M, Tomino Y, Koide H. Effectof a specific endothelin receptor A antagonist on glomeru-lonephritis of ddY mice with I gA nephropathy. Nephron.1996;72:454-460.14. Liao J, Hayashi K, Horikoshi S, Ushijima H, Kimura J,Tomino Y. Effect of steroid-liposome on immunohistopathol-ogy of I gA nephropathy in ddY mice. Nephron.2001;89:194-200.Received: September 21, 2007Accepted: November 04, 2007© Società Italiana di Nefrologia467JN EPHROL2008; 21: 463-467。
一种IgA肾病小鼠肾功能不全模型的建立方法[发明专利]
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(19)中华人民共和国国家知识产权局(12)发明专利申请(10)申请公布号 (43)申请公布日 (21)申请号 201810844253.5(22)申请日 2018.07.27(71)申请人 上海中医药大学附属曙光医院地址 200021 上海市黄浦区普安路185号(72)发明人 高建东 史丽强 万强 刘伟伟 吴燕升 林评兰 (74)专利代理机构 上海卓阳知识产权代理事务所(普通合伙) 31262代理人 周春洪(51)Int.Cl.A01K 67/02(2006.01)A61K 35/74(2015.01)A61K 38/38(2006.01)A61K 49/00(2006.01)(54)发明名称一种IgA肾病小鼠肾功能不全模型的建立方法(57)摘要本发明涉及一种IgA肾病小鼠肾功能不全模型的建立方法，所述方法选取BALB/C小鼠为动物模型，实验步骤为：1.隔日给予口服低内毒素牛血清白蛋白酸化水溶液灌胃，持续五周；2.第六周时定时尾静脉注射2％BSA缓冲液0.2mL，每日1次，连续3天；3.第9周至第11周观察小鼠一般状态；4.第12周起尾静脉注射SEB缓冲液0.2mL，剂量为每只小鼠0.8mg/kg，每周1次，连续3周；第15周为试验终点。
其优点表现在：(1)本发明有效降低了IgA肾病小鼠的死亡率；(2)本模型可以更好的模拟人类IgA肾病的发病机制，进一步筛选治疗IgA肾病的药物。
(3)技术操作简便，动物模型成功率高，有很好的应用前景。
权利要求书2页 说明书7页 附图3页CN 108935316 A 2018.12.07C N 108935316A1.一种IgA肾病小鼠肾功能不全模型，是由下列方法制备得到，该方法选取BALB/C小鼠为动物模型，所述方法包含以下步骤：(1)选取BALB/C小鼠适应性喂养一周后，隔日给予口服低内毒素牛血清白蛋白酸化水溶液灌胃，持续五周；(2)将步骤1所得到的小鼠在第六周时定时尾静脉注射2％BSA缓冲液0.2mL，每日1次，连续3天；(3)第9周至第11周观察小鼠一般状态；(4)第12周起尾静脉注射SEB缓冲液0.2mL，剂量为每只小鼠0.8mg/kg，每周1次，连续3周；(5)第15周为试验终点。
关于肾病小鼠模型创建的研究进展
关于肾病小鼠模型创建的研究进展引言肾脏疾病是全球范围内导致死亡和病残的主要原因之一。
为了更好地理解和治疗肾脏疾病，科学家们一直致力于肾病小鼠模型的创建和研究。
本文将介绍肾病小鼠模型创建的研究进展。
1. 肾脏疾病的重要性肾脏是人体的重要器官，负责排除废物和调节体内水平衡。
肾脏疾病包括慢性肾病、肾衰竭和肾癌等，对患者的生活质量和寿命造成了严重的影响。
因此，研究肾脏疾病的发病机制和寻找治疗方法具有重要意义。
2. 肾病小鼠模型的创建方法创建肾病小鼠模型是研究肾脏疾病的关键一步。
目前常用的肾病小鼠模型创建方法主要包括药物诱导、基因敲除和基因突变等。
- 药物诱导：通过注射特定的药物，如阿米洛利德(Amiloride)、环磷酰胺(Cyclophosphamide)等，诱导小鼠出现肾脏病理变化，模拟人类肾脏疾病的发生过程。
- 基因敲除：通过特定的基因敲除技术，使小鼠的基因发生缺陷，进而导致肾脏病变。
- 基因突变：通过诱导基因突变，使小鼠产生与人类肾脏疾病相似的表型。
3. 肾病小鼠模型在研究中的应用肾病小鼠模型在肾脏疾病的研究中发挥着重要作用。
- 发病机制研究：通过肾病小鼠模型，研究者可以深入了解肾脏疾病的发病机制，如肾小球肾炎的发生、肾肿瘤的生成等。
- 药物筛选和治疗评估：通过肾病小鼠模型，可以对新药进行评估和筛选，寻找治疗肾脏疾病的有效方法。
- 基因治疗研究：通过肾病小鼠模型，可以研究基因治疗对于肾脏疾病的疗效和安全性，为基因治疗的临床应用提供参考。
4. 近年来的研究进展近年来，肾病小鼠模型的创建和研究取得了一些重要的进展。
- 利用基因编辑技术：利用CRISPR/Cas9等基因编辑技术，科学家们成功地创建了一系列与肾脏疾病相关的基因突变小鼠模型，如肾小管上皮细胞特异性基因敲除小鼠、肾小球特异性基因突变小鼠等。
- 结合转基因技术：通过结合转基因技术和药物诱导等方法，科学家们成功地创建了一些肾脏病变特异性的小鼠模型，如IgA肾病小鼠模型、糖尿病肾病小鼠模型等。
IgA-Ⅰ号对IgA肾病小鼠肾及肝脾肺组织病理的影响
四味 片 组 ， 另设 正 常组 ， 组 １ 。治 疗 ４周 后 处 死全 部 小 鼠 ， 双 肾及 部 分 肝 、 、 组 织 进 行 组 织病 理 检 查 。 结 果 ： １ 并 每 ０只 取 脾 肺 （）
模型组的免 疫荧光 ： Ａ表达很 强， 和 ｃ ｌ ｅ Ｉ ３次之 ； 光镜 ： 肾小球 系膜细胞和基质增生 ； 电镜 ： 系膜 区可见电子致密物沉积 ， 表
材 料 与方 法
９ ％ ，０ ％） ３次 ， ％ 锇 酸 磷 酸 缓 冲 液 固 定 ２ ， 氧 树 脂 ０ １０ 各 １ ４ｈ环 Ｅ ｏ １ 理 ， 薄 切 片 机 切 片 ， 酸 双 氧 铀 枸 椽 酸 铝 电 子 染 ｐｎ８ ２包 超 醋 色 ， Ｍ 一１０ Ｊ Ｅ ２０电子 显 微 镜下 观察 。 ４ 主 要 试 剂 及 药 品 兔 抗 鼠 ｌ ， ３ 克 隆 抗 体 （ ａｔ Ａ ｅ ｃ单 Ｓ ｎａ
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高强度沉积 的一组临床综合 征。发病机制 尚不完全清楚 ， 可供 切 片 ， 制成 ４ｐｎ厚 的 冰冻 切 片 ， 丙 酮 固定 ２ ｉ， 接 免 ｚ 纯 ～３ｒ ｎ 直 ａ
［ 摘 要］ 目的 ： 观察 ＩＡ— Ｉ ｇ 号对 ＩＡ肾病小鼠 肾脏及肝 、 肺组织病理 的影响 。方法 ： ｇ 脾、 用金 黄 色葡萄球 菌细胞膜 ２ ０
肽 抗 原 决 定簇 （０ ］ Ａ 诱 导 Ｉ 肾病 模 型 。病 理 鉴 定模 型 成 功 后 ， 机 分 为 模 型 组 、 ２Ｐ ｓ ） Ａ ｅ 随 Ｔ号 高 剂 量 组 、 Ｉ号低 剂 量 组 、 炎 肾
I_A_1基因表达与小鼠膜性肾小球肾炎
I-A β1基因表达与小鼠膜性肾小球肾炎张旭，高岑，余绍兰，马跃荣（泸州医学院病理教研室，四川泸州646000）摘要目的：探讨I-A β1基因与实验性小鼠膜性肾小球肾炎发病之间的相关性。
方法:复制并鉴定小鼠膜性肾小球肾炎动物模型，提取实验组和对照组总RNA ，实时荧光定量PCR 检测I-A β1基因mRNA 表达量。
结果：模型组I-A β1基因mR -NA 表达量明显高于N-BSA 组、NS 组和空白对照组(P<0.01)差异有统计学意义；N-BSA 组、NS 组和空白对照组彼此之间的差异无统计学意义。
结论：I-A β1基因过量表达参与了实验性小鼠膜性肾小球肾炎发病过程，可能是通过I-A β1基因转录、翻译活性上调使小鼠对外源性抗原反应性增强，促进了小鼠膜性肾小球肾炎发生。
关键词I-A β1；实时荧光定量PCR ；膜性肾小球肾炎中图分类号R363.21文献标识码A文章编号1000-2669（2009）3-0218-04I-A β1GENE EXPRESSION AND MOUSE MEMBRANOUSGLOMERULONEPHRITISZhang Xu,et alDepartment of Pathology,Luzhou Medical CollegeAbstract Objective:To study the relationship between I -A β1gene and the pathogenesis of membranous glomerulonephritis in experimental mouse.Methods:The animal model of MGN was reproduced and identified in mouse,then the total RNA of experimental group and control group was extracted.After I-A β1gene was extract -ed,spleen I -A β1mRNA expression was detected by real time fluorescent quantitation PCR.Results:I -A β1gene mRNA expression in model group was remarkably higher than that in group N-BSA,group NS and blank group (P<0.01),but there wasn ’t statistic significance among group N-BSA,group NS and blank group.Conclu -sion:I-A β1gene overexpression may participate in the pathogenetic process of MGN in experimental mice;its potential nosogenesis is the up-regulation of I-A β1genetic transcription and translation to promote antigen-reac -tive ability and morbility of MGN.Key wordsI-A β1;Real time fluorescent quantitative PCR;MGN作者简介：张旭（1977-），男，讲师，硕士膜性肾小球肾炎(membranous glomerulonephri -tis,MGN)是引起成人肾病综合征最常见的原因，多数患者预后差。
IgA肾病风险预测模型建立及验证演示稿件
该文献对IgA肾病风险预测模型的建 立和验证进行了系统评价，为模型的 优化和改进提供了依据。
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IgA肾病风险预测模 型建立及验证
目 录
• 引言 • IgA肾病概述 • 风险预测模型建立 • 风险预测模型验证 • 讨论与展望 • 参考文献
01
CATALOGUE
引言
研究背景
IgA肾病是常见的原发性肾小球疾病 ，其发病机制复杂，且个体差异大， 导致临床诊断和治疗难度较大。
早期预测和干预对于降低IgA肾病进展 风险具有重要意义，因此建立有效的 风险预测模型是当前研究的热点。
研究目的和意义
目的
本研究旨在建立一种能够准确预测IgA肾病风险的预测模型， 为临床医生提供决策支持，帮助患者早期发现和治疗，降低 疾病进展风险。
意义
通过建立有效的风险预测模型，可以实现对IgA肾病的高危人 群进行早期筛查和干预，提高患者的生活质量和预后。同时 ，该模型也可以为其他肾小球疾病的预测提供借鉴和参考， 推动肾脏病领域的发展。
模型训练
使用训练数据集对模型进行训练，调整模型参数，提高模型的预测 精度。
模型优化
采用集成学习、超参数调整等方法对模型进行优化，提高模型的泛化 能力。
04
CATALOGUE
风险预测模型验证
验证方法
1 2
模型构建
基于临床数据和生物学指标，构建IgA肾病风险 预测模型。
数据集划分
将数据集划分为训练集和验证集，用于训练和验 证模型。
患者可以了解自己的风险状况，采取 相应的预防措施，改善生活习惯，降 低疾病发生的风险。
辅助诊断
个体化治疗
预后评估
健康管理
IgA肾病风险预测模型能够帮助医生 在患者发病早期识别高风险人群，为 早期干预和预防提供依据。
益肾汤对实验性IgA肾病模型小鼠肾组织转化生长因子β1表达的影响
益肾汤对实验性IgA肾病模型小鼠肾组织转化生长因子β1表达的影响作者：万启军，何永成，石成钢，洪国保，胡斌，栾韶东【摘要】【目的】观察益肾汤对实验性IgA肾病（IgAN）模型小鼠肾组织转化生长因子β1（TGFβ1）表达的影响。
【方法】选用BALB/c小鼠，采用口服牛血清白蛋白联合尾静脉注射葡萄球菌肠毒素B方法复制小鼠IgAN模型，抽样检测造模小鼠24h尿蛋白定量、肾组织过碘酸雪夫反应（PAS）染色及免疫荧光检查，确定造模成功后随机分为模型组，益肾汤高、低剂量组（剂量分别为01、005g·kg-1·d-1），并设正常对照组；采用荧光定量聚合酶链反应（FQ PCR）法检测各组小鼠肾组织TGFβ1mRNA表达，免疫组化法检测各组肾组织TGFβ1含量。
【结果】模型组肾组织TGFβ1mRNA表达及TGFβ1含量均显著升高（Ｐ＜００５）；益肾汤高、低剂量组肾组织TGFβ1mRNA表达及TGFβ1含量均显著降低（Ｐ＜００５）。
【结论】益肾汤治疗IgAN的作用与抑制肾组织中TGFβ1的分泌及其mRNA表达有关。
【关键词】益肾汤/药理学；IgA肾病/中药疗法；肾/病理学；转化生长因子β；疾病模型，动物；小鼠研究证实细胞因子在IgA肾病（IgAN）的发生发展过程中起重要作用，其中，转化生长因子β1（TGFβ1）是公认的最重要的致纤维化因子［1］。
本研究观察了TGFβ1在实验性IgAN小鼠肾组织中表达的特点及中药方剂益肾汤对TGFβ1的影响，现报道如下。
1材料与方法11益肾汤组方及制备益肾汤组成：黄芪15g、生地20g、牡丹皮10g、熟地15g、赤芍10g、当归10g、山药15g、茯苓15g、山茱萸15g、金樱子15g、枸杞子15g、白茅根30g、女贞子15g、旱莲草15g、仙鹤草20g。
按照“人—鼠用药剂量转换公式”计算，浓缩成高浓度（相当于成人剂量的2倍）及低浓度（相当于成人的每日用量）2种剂型，原液装瓶，高压消毒后密封保存，备用。
改良大鼠IgA肾病模型建立及氧化应激状态评估
改良大鼠IgA肾病模型建立及氧化应激状态评估高明;张连栋;张莉;王昌;卢燕【摘要】Objective To modify the traditional IgA nephropathy model and evaluate the oxidative stress status of the model,providing experimental evidence for the establishment of IgA nephropathy animal model.Methods Twelve female SPF SD rats weighing 200-240 g were randomly divided into normal control group (group C) and modified model group (group M) according to the random digital table method.The animals in group C were gavaged with normal saline at 4 ml/kg and given subcutaneous injection at 0.4 ml,and those in group M were treated with bovine serum albumin at 600 mg/kg every day by gavage,and subcutaneously injected with carbon tetrachloride at 0.10 ml,castor oil at 0.3 ml,thymosin at 3 mg and LPS at 0.05 mg.At the 12th week,the general states of rats in two groups were observed.Serum creatinine (SCr),blood urea nitrogen (BUN),albumin (Alb) and 24-h urine protein were determined.IgA immunofluorescence,HE staining,biochemical index as well as the state of oxidative stress in the kidney were evaluated.Results All the rats in the group M suffered from mental depression,tiredness,decreased movement and sparse hair.BUN and SCr levels in group M were significantly higher (P＜0.05),the level of Alb was significantly lower (P＜0.01),and 24-h urine protein was significantly higher than those in group C (P＜0.05).In group M,IgA clumps were deposited in the renal mesangial region,the glomerular mesangial area was slightly widened,even moderately broadened,and increasedmesangial cells and matrix were observed by HE staining.The levels of SOD and T-AOC in renal tissue of group M were significantly lower (P ＜0.05),and the level of MDA was significantly higher than that of group C (P ＜ 0.05).Conclusions The modified IgA nephropathy rat model is optimal,and the biochemical and pathological indexes are sihailar to those of human IgA nephropathy.The change of oxidative stress in the kidney is one of the important pathological mechanisms involved in the process.%目的对原有IgA肾病(IgAN)模型进行改进,并评估该模型肾脏氧化应激状态改变,为探索IgAN模型建立方法提供实验依据.方法取SPF级SD雌性大鼠12只,体质量200～240 g,采用随机数字表法随机分为正常对照组(C组)及改良模型组(M组).C组生理盐水4 ml/kg灌胃,皮下注射生理盐水0.4 ml;M组采用牛血清白蛋白600 mg/kg隔日灌胃+皮下注射四氯化碳0.10 ml、蓖麻油0.3 ml及胸腺肽3 mg+尾静脉注射脂多糖0.05 mg建立模型,观察至第12周末,分别观察2组大鼠一般状态、检测2组血肌酐(SCr)、尿素氮(BUN)、白蛋白(Alb)并测定24h尿蛋白定量,肾脏组织行IgA免疫荧光、苏木素-伊红(HE)染色并评估肾脏组织氧化应激状态.结果M组大鼠均出现精神萎靡、倦卧少动、毛发稀疏等状况.M组BUN、SCr较C组显著升高(P＜0.05),Alb水平较C组显著降低(P＜0.01);24h尿蛋白定量较C组显著升高(P＜0.05).M组大鼠肾脏系膜区IgA团块样沉积,HE染色M组可见肾小球系膜区轻度增宽,偶见中度增宽,系膜细胞及基质增多.M组肾脏组织超氧化物歧化酶、总抗氧化能力水平较C组均显著下降(P＜0.05),丙二醛水平显著高于C组(P＜0.05).结论改良大鼠IgAN模型造模效果良好,生化及病理指标与人IgAN类似,肾脏氧化应激状态改变是该过程中重要的机制之一.【期刊名称】《临床肾脏病杂志》【年(卷),期】2018(018)004【总页数】5页(P243-247)【关键词】IgA肾病;动物模型;氧化应激【作者】高明;张连栋;张莉;王昌;卢燕【作者单位】710003 西安市第四医院肾内科;710004 西安交通大学第二附属医院泌尿外科;710003 西安市第四医院肾内科;710003 西安市第四医院肾内科;解放军第306医院航天城门诊部【正文语种】中文IgA 肾病(IgAN)居原发性肾小球疾病首位，临床表现多样，主要临床表现是血尿，部分患者可伴发蛋白尿，是导致终末期肾病(ESRD)的重要原因之一[1]。
IgA肾病小鼠模型的建立与鉴定
IgA肾病小鼠模型的建立与鉴定【摘要】目的建立小鼠IgA肾病模型，为临床研究IgA肾病提供实验依据。
方法昆明种小鼠30只，分为正常对照组(10只)、模型组(20只)。
采用口服牛血清白蛋白(BSA)联合尾静脉注射葡萄球菌肠毒素B(SEB)的方法制作小鼠IgA肾病模型。
第12周末收集新鲜尿液，显微镜下尿红细胞定性检验;摘眼球取血，检测血肌酐(Scr)、尿素氮(BUN);光镜法观察肾小球系膜细胞及基质变化;电镜法检测系膜区有无电子致密物沉积;免疫荧光法观察系膜区IgA沉积情况。
结果第12周末，模型组小鼠均出现不同程度的血尿;Scr、BUN较正常对照组显著增高(P<0.01);光镜下系膜细胞及基质明显增生;电镜下系膜区电子致密物沉积;免疫荧光示系膜区大量的IgA沉积，而正常对照组则无上述表现。
结论 IgA肾病小鼠模型效果良好，病理、电镜及临床指标与人类IgA肾病临床病理相似。
【关键词】 IgA肾病;模型;血尿;免疫荧光;小鼠Abstract: Objective To establish mouse model of IgA nephropathy in order to provide experiment basis for the clinical research of IgA nephropathy. Methods 30 Kunming mice were randomly assigned into the control group (n=10) and the model group (n=20). Mouse model of IgA nephropathy was established by the method of oral intake of bovine serum albumin (BSA) together with injection of staphylococcus enterotoxin B (SEB) via the caudal vein. At the end of the 12 weeks, fresh urinewas gathered for the quantitative test of hematuria; blood was taken through orbital sinus after eyeball extirpation to determine the serum creatinine (Scr) and blood urea nitrogen (BUN); the changes of mesangial cells and matrix in the kidneys were observed by light microscopy; electron microscopy was employed to detect electron-dense deposits in the mesangial area; the expression of IgA deposition was measured by immunofluorescence staining in the mesangial area. Results By the end of 12 weeks, there were different degrees of hematuria in model group mice. BUN and Scr levels in model group were significantly higher than in the normal control group (P<0.01). Light microscopy showed evident proliferation of mesangial cells and matrix. Electron microscopy demonstrated a high electron dense deposition in the mesangial area, while the above abnormalities were not observed in the normal control group.Conclusion The mouse model of IgA nephropathy proves effective, with pathological electron microscopic and clinical parameters similar to the clinical pathology of human IgA nephropathy.Key words: IgA nephropathy; model; hematuria; immunofluoresence staining; miceIgA肾病世界范围内广发的一种系膜增生型肾小球肾炎，占原发性肾小球疾病的10%～20%，中国、日本、新加坡等发病率较高，其中有20%～30%的患者进展到肾衰[1]。
大鼠IgA肾病模型的建立及血清中IL-6、FN、NO的检测
大鼠IgA肾病模型的建立及血清中IL-6、FN、NO的检测刘小平;胡顺金;方琦【期刊名称】《中国比较医学杂志》【年(卷),期】2008(18)1【摘要】目的建立大鼠IgA肾病(IgAN)模型,测定大鼠血清中的白介素-6(IL-6)、纤维结合蛋白(FN)、一氧化氮(NO),探讨这些指标水平的变化与IgAN免疫损伤的相关性,为临床治疗提供动物实验研究依据.方法 24只大鼠被随机分成3组,每组8只.模型组和治疗组用免疫复合物法复制;正常对照组用生理盐水.10周后,治疗组大鼠被给予雷公藤多甙片3周.留取所有大鼠血清测定IL-6、FN、NO;留取所有大鼠尿液测定红细胞(RBC)、总蛋白量(TPR);留取所有大鼠肾组织作病理学检查.结果模型组中大鼠尿液中RBC、TPR含量较治疗组及正常对照组显著增高(P ＜ 0.01);血清中的IL-6、FN的水平较治疗组及正常对照组显著增高(P ＜ 0.01);血清中的NO 水平较治疗组及正常对照组显著降低(P ＜ 0.01).治疗组的大鼠肾组织病理损伤程度较模型组明显减轻(P ＜ 0.01).结论血清中的IL-6、FN及NO的水平与RBC数、TPR及肾组织病理损伤程度相关,它们可作为观察IgAN治疗效果的重要指标,也可作为IgAN严重性的预测指标.下调血中的IL-6、FN及上调NO的水平,可减少IgA与FN免疫复合物的形成,从而改善肾组织的免疫损伤.【总页数】5页(P5-8,插2)【作者】刘小平;胡顺金;方琦【作者单位】安徽中医学院第一附属医院,合肥,230031;安徽中医学院第一附属医院,合肥,230031;安徽中医学院第一附属医院,合肥,230031【正文语种】中文【中图分类】R332【相关文献】1.血清IgA-FN、Ⅳ型胶原在IgA肾病患者中的临床意义 [J], 李贞琼;王玉梅;刘建社;邓安国2.健肾片对大鼠实验性IgA肾病模型血清IL-2、IL-6含量的影响 [J], 孙伟;曾安平;王钢;易岚;盛梅笑;何伟明;周恩超3.青蒿琥酯对IgA肾病模型大鼠血清IL-2、IL-6含量的影响 [J], 马云霞;栾森;马行一;曾燕;吴蔚桦;米绪华;秦伟4.IgA肾病大鼠模型血清IL-2和IL-6变化对肾小管上皮细胞TGF-β1表达的影响[J], 冷斌;尹友生;韦家智;肖敏5.补肾健脾、利湿化瘀法对IgA肾病模型大鼠血清TNF-α、IL-6含量的影响 [J], 张守琳因版权原因，仅展示原文概要，查看原文内容请购买。
人类肾脏疾病动物模型研究进展
作者简介：王倩茹，在读硕士研究生；研究方向：分子药理学；Ｅ ｍａｉｌ：ｗｑｒ９８１０２０＠１６３．ｃｏｍ侯久文为本文共同第一作者，贡献与第一作者同等通讯作者：梅其炳，教授，博士生导师；研究方向：分子药理学；Ｅ ｍａｉｌ：ｑｂｍｅｉ＠ｓｗｍｕ．ｅｄｕ．ｃｎ人类肾脏疾病动物模型研究进展王倩茹１，２ 侯久文１，２ 梅其炳１，２１．西南医科大学药学院，泸州，６４６０００，中国２．泸州新药评价研究中心，泸州，６４６０００，中国【摘要】 肾脏疾病严重威胁人们健康，构建与人类肾脏疾病更加相近的动物模型对深入探讨肾脏疾病的发病机制，以及发现治疗药物具有十分重要意义。
该文根据肾脏疾病诱因对动物模型进行分类，分别对糖尿病肾病、肾小球肾炎、高血压肾病、尿酸性肾病、梗阻性肾病、药物肾损伤性及缺血性肾病的实验动物模型及其研究进展进行综述。
【关键词】 动物模型；肾脏疾病；人类疾病动物模型【中图分类号】 Ｒ９６５．１ 【文献标识码】 Ａ 犇犗犐：１０．３９６９／ｊ．ｉｓｓｎ．２０９５ １３９６．２０２３．０４．００５犚犲狊犲犪狉犮犺犘狉狅犵狉犲狊狊狅狀犃狀犻犿犪犾犕狅犱犲犾狊狅犳犎狌犿犪狀犓犻犱狀犲狔犇犻狊犲犪狊犲ＷＡＮＧＱｉａｎ ｒｕ１，２，ＨＯＵＪｉｕ ｗｅｎ１，２，ＭＥＩＱｉ ｂｉｎｇ１，２１．ＣｏｌｌｅｇｅｏｆＰｈａｒｍａｃｙ，ＳｏｕｔｈｗｅｓｔＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｌｕｚｈｏｕ，６４６０００，Ｃｈｉｎａ２．ＬｕｚｈｏｕＮｅｗｄｒｕｇＥｖａｌｕａｔｉｏｎＲｅｓｅａｒｃｈＣｅｎｔｅｒ，Ｌｕｚｈｏｕ，６４６０００，Ｃｈｉｎａ【犃犅犛犜犚犃犆犜】 Ｋｉｄｎｅｙｄｉｓｅａｓｅｉｓａｓｅｒｉｏｕｓｔｈｒｅａｔｔｏｐｅｏｐｌｅ’ｓｈｅａｌｔｈ，ｓｏｉｔｉｓｖｅｒｙｉｍｐｏｒｔａｎｔｔｏｒｅｐｌｉｃａｔｅｔｈｅａｎｉｍａｌｍｏｄｅｌｗｈｉｃｈｉｓｍｏｒｅｓｉｍｉｌａｒｔｏｈｕｍａｎｋｉｄｎｅｙｄｉｓｅａｓｅｉｎｏｒｄｅｒｔｏｆｕｒ ｔｈｅｒｅｘｐｌｏｒｅｔｈｅｐａｔｈｏｇｅｎｅｓｉｓｏｆｋｉｄｎｅｙｄｉｓｅａｓｅａｎｄｆｉｎｄｔｈｅｒａｐｅｕｔｉｃｄｒｕｇｓ．Ｉｎｔｈｉｓｐａｐｅｒ，ａｎｉ ｍａｌｍｏｄｅｌｓｏｆｄｉａｂｅｔｉｃｎｅｐｈｒｏｐａｔｈｙ，ｇｌｏｍｅｒｕｌｏｎｅｐｈｒｉｔｉｓ，ｈｙｐｅｒｔｅｎｓｉｖｅｎｅｐｈｒｏｐａｔｈｙ，ｕｒｉｃａｃｉｄｎｅｐｈｒｏｐａｔｈｙ，ｏｂｓｔｒｕｃｔｉｖｅｎｅｐｈｒｏｐａｔｈｙ，ｄｒｕｇ ｉｎｄｕｃｅｄｒｅｎａｌｉｎｊｕｒｙａｎｄｉｓｃｈｅｍｉｃｎｅｐｈｒｏｐａｔｈｙｗｅｒｅｒｅｖｉｅｗｅｄ．【犓犈犢犠犗犚犇犛】 ａｎｉｍａｌｍｏｄｅｌ；ｋｉｄｎｅｙｄｉｓｅａｓｅ；ａｎｉｍａｌｍｏｄｅｌｏｆｈｕｍａｎｄｉｓｅａｓｅ 肾脏疾病可根据血清肌酐浓度或肾小球滤过率（ｇｌｏｍｅｒｕｌａｒｆｉｌｔｒａｔｉｏｎｒａｔｅ，ＧＦＲ）及持续时间分为急性肾损伤（ａｃｕｔｅｋｉｄｎｅｙｉｎｊｕｒｙ，ＡＫＩ）和慢性肾病（ｃｈｒｏｎｉｃｋｉｄｎｅｙｄｉｓｅａｓｅ，ＣＫＤ）。
自发IgA肾病的ddY小鼠模型研究进展
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自发 ＩｇＡ肾病 的 ｄｄＹ小 鼠模 型研 究 进展
刘伟 伟① 万 强① 吴燕升① 史丽 强① 黄 迪① 高建 东①
Ｘｇａ 肾病 （Ｉｍｍｕｎｏｇｌｏｂｕｌｉｎ ＩｇＡ ｎｅｐｈｒｏｐａｔｈｙ，ｔｇＡＮ）是 全球 最 ７６％ 的 ＨＩＧＡ 小 鼠（１６／２１）肾脏 出现 中到 重 度 的 系 膜 增 生 ，
机 制已经建 立 了多 种 动物 模 型 ，其 中 自发 类 动物 模 型一ｄｄＹ 交 ，从 第二 代开始 是近亲 繁殖 ，暗示我 们高 血清 ＩｇＡ小 鼠可能 （Ｄｅｕｔｓｃｈｌａｎｄ，Ｄｅｎｋｅｎ，Ｙｏｋｅｎ）小 鼠在 疾病 发生 、发 展等 方 面与 存在某些未知 的基 因与血清 ＩｇＡ有关 。后来被证 实 １２号染色
人类 ＩｇＡＮ极为相似 ，引起 了科研 工作 者的关 注。本文 拟对 可 体上 的 Ｄ１２Ｍｉ￣０与血清 ＩｇＸ水 平有关 。但是 该模型仅 少数
自发 ＩｇＡＮ的 ｄｄＹ小 鼠动物模 型的研究进 展作 一综 述。
的 ＨＩＧＡ小 鼠出现 了蛋 白尿 （１００～３００ ｍｇ／ｄ１），全程未见血 尿 ，
血清 ＩｇＡ小 鼠 （ｈｉｇｈ ｓｅｌ＇ｔｌｍ ＩｓＡ ｍｉｃｅ，ＨＩＧＡ小 鼠 ），ｇｄｄＹ小 鼠 现了系膜增生伴严重 的肾小球和 肾小管 间质的损伤 ， 肾小 球系
（ｇｒｏｕｐｅｄ ｄｄＹ ｍｉｃｅ）。２００５年 Ｓｕｚｕｋｉ等 通过全基 因组 扫描技 膜扩张 ，肾间质浸润 ；在 ８周龄 和 ２４周 龄 ，ｇｄｄＹ小 鼠肾小球 细
１ ｄｄＹ小 鼠简 介
与人类 ＩｇＡＮ临床表现 不符 。 ２．３ ｇｄｄＹ小 鼠 ｄｄＹ小 鼠属 于 远 交 系 ，因 此 导 致 发
BSA+SEB制作小鼠IgA肾病模型的注意事项
的ＲＳＤ＜２％㊂检测过程中，热处理室表面温度可达１００ħ以上，注意不要触摸，避免烫伤㊂石英舟㊁起泡器等附件均为易碎品，且价格较高，操作过程中注意避免掉落㊂汞分析仪也可用于空气中超低浓度汞含量的实时监测，还可用于水㊁土壤等样品中汞的检测，在国内外均有应用［１６－１７］㊂该方法操作简便㊁速度快㊁检测费用低，可用于大批量快速筛查㊂参考文献：［１］㊀胡月红．国内外汞污染分布状况研究综述［Ｊ］．环境保护科学，２００８，３４（１）：３８－４１．［２］㊀刘思妹，朱毅，郝睿．国内外汞污染现状及管理措施［Ｊ］．环境科学与技术，２０１４，３７（Ｓ２）：２９０－２９４．［３］㊀孙贵范．职业卫生与职业医学［Ｍ］．第７版．北京：人民卫生出版社，２０１２：７７－７８．［４］㊀姜枫，蒋莹，王军明．职业接触者尿中汞微波消解⁃原子荧光法测定［Ｊ］．中国公共卫生，２０１５，３１（１２）：１６２９－１６３１．［５］㊀ＳｈａｈＡＱ，ＫａｚｉＴＧ，ＢａｉｇＪＡ，ｅｔａｌ．Ｓｉｍｕｌｔａｎｅｏｕｓｌｙｄｅｔｅｒｍｉｎａｔｉｏｎｏｆｍｅｔｈｙｌａｎｄｉｎｏｒｇａｎｉｃｍｅｒｃｕｒｙｉｎｆｉｓｈｓｐｅｃｉｅｓｂｙｃｏｌｄｖａｐｏｒｇｅｎｅｒａｔｉｏｎａｔｏｍｉｃａｂｓｏｒｐｔｉｏｎｓｐｅｃｔｒｏｍｅｔｒｙ［Ｊ］．ＦｏｏｄＣｈｅｍ，２０１２，１３４（４）：２３４５－２３４９．［６］㊀ＨｕｂｅｒＪ，ＨｅｉｍｂüｒｇｅｒＬＥ，ＳｏｎｋｅＪＥ，ｅｔａｌ．Ｎａｎｏｇｏｌｄ⁃ｄｅｃｏｒａｔｅｄｓｉｌｉｃａｍｏｎｏｌｉｔｈｓａｓｈｉｇｈｌｙｅｆｆｉｃｉｅｎｔｓｏｌｉｄ⁃ｐｈａｓｅａｄｓｏｒｂｅｎｔｆｏｒｕｌｔｒａｔｒａｃｅｍｅｒｃｕｒｙａｎａｌｙｓｉｓｉｎｎａｔｕｒａｌｗａｔｅｒｓ［Ｊ］．ＡｎａｌＣｈｅｍ，２０１５，８７（２１）：１１１２２－１１１２９．［７］㊀李明章，林建奇．微波消解⁃原子荧光光谱法同时测定食醋中的砷和汞［Ｊ］．理化检验⁃化学分册，２０１６，５２（２）：２２２－２２５．［８］㊀杨常青，张双双，李芳，等．氧弹分解⁃原子荧光法快速测定煤中汞［Ｊ］．分析试验室，２０１６，３５（５）：５６５－５６７．［９］㊀ｄｅＳｏｕｚａＳＳ，ＣａｍｐｉｇｌｉａＡＤ，ＢａｒｂｏｓａＦＪｒ．Ａｓｉｍｐｌｅｍｅｔｈｏｄｆｏｒｍｅｔｈｙｌｍｅｒｃｕｒｙ，ｉｎｏｒｇａｎｉｃｍｅｒｃｕｒｙａｎｄｅｔｈｙｌｍｅｒｃｕｒｙｄｅｔｅｒｍｉｎａｔｉｏｎｉｎｐｌａｓｍａｓａｍｐｌｅｓｂｙｈｉｇｈｐｅｒｆｏｒｍａｎｃｅｌｉｑｕｉｄｃｈｒｏｍａｔｏｇｒａｐｈｙ⁃ｃｏｌｄ⁃ｖａｐｏｒ⁃ｉｎｄｕｃｔｉｖｅｌｙｃｏｕｐｌｅｄｐｌａｓｍａｍａｓｓｓｐｅｃｔｒｏｍｅｔｒｙ［Ｊ］．ＡｎａｌＣｈｉｍＡｃｔａ，２０１３，７６１：１１－１７．［１０］㊀袁利杰，李云，郑子栋，等．微波消解⁃电感耦合等离子质谱法测定龟甲胶中６种有害元素［Ｊ］．分析科学学报，２０１４，３０（４）：５７８－５８０．［１１］㊀ＧＢ７４６９－８７．总汞的测定，双硫腙分光光度法［Ｓ］．［１２］㊀王斌，况丽，钟康华，等．乙二胺四乙酸二钠络合消解⁃电感耦合等离子体原子发射光谱法测定塑料中的铅㊁镉㊁铬和汞［Ｊ］．理化检验⁃化学分册，２０１６，５２（５）：５５２－５５４．［１３］㊀宋永刚，于彩芬，张玉凤，等．直接测汞仪与原子荧光法测定海洋底栖生物中痕量汞的对比研究［Ｊ］．分析科学学报，２０１６，３２（２）：２８８－２９０．［１４］㊀ＷＳ／Ｔ２６－１９９６．汞的测定，冷原子吸收光谱法，Ⅱ酸性氯化亚锡还原法［Ｓ］．［１５］㊀ＧＢＺ／Ｔ２１０ ５－２００８．汞的测定，生物材料中化学物质测定方法［Ｓ］．［１６］㊀周巧丽，郭鹏然，潘佳钏，等．活性炭富集⁃电热塞曼原子吸收光谱法测定水中痕量的汞［Ｊ］．分析化学，２０１６，４４（８）：１２７０－１２７６．［１７］㊀ＤｉＮａｔａｌｅＦ，ＥｒｔｏＡ，ＬａｎｃｉａＡ，ｅｔａｌ．Ｍｅｒｃｕｒｙａｄｓｏｒｐｔｉｏｎｏｎｇｒａｎｕｌａｒａｃｔｉｖａｔｅｄｃａｒｂｏｎｉｎａｑｕｅｏｕｓｓｏｌｕｔｉｏｎｓｃｏｎｔａｉｎｉｎｇｎｉｔｒａｔｅｓａｎｄｃｈｌｏｒｉｄｅｓ［Ｊ］．ＪＨａｚａｒｄＭａｔｅｒ，２０１１，１９２（３）：１８４２－１８５０．收稿日期 ２０１７－０６－１３㊃专家问答㊃问：ＢＳＡ＋ＳＥＢ制作小鼠ＩｇＡ肾病模型的注意事项答：国内多采用口服ＢＳＡ＋尾静脉注射ＳＥＢ的方法来制作小鼠ＩｇＡ肾病模型，根据文献及我们的研究，这种方法成模死亡率高，死亡率可达到７０％左右，在多次重复实验中我们得出如下经验：（１）采用ＢＡＬＢ／ｃ小鼠㊂我们在实验中发现昆明小鼠死亡率显著高于ＢＡＬＢ／ｃ小鼠，ＢＡＬＢ／ｃ小鼠的死亡率为４０％左右，昆明小鼠死亡率约为７０％，ＢＡＬＢ／ｃ小鼠经常在免疫学研究中使用，抗过敏能力强，有文献报道与ＴＨ２免疫失衡有关㊂（２）增加保温措施㊂小鼠在注射ＢＳＡ后会蜷缩在一起，体表温度下降，给予保温措施可降低小鼠死亡可能性㊂（３）采用低内毒素ＢＳＡ㊂目前ＢＳＡ大致分为标准级ＢＳＡ㊁ＢＳＡ第五组分㊁无ＩｇＧ试剂级ＢＳＡ㊁无蛋白酶ＢＳＡ㊁无脂肪酸ＢＳＡ㊁低内毒素ＢＳＡ㊁无蛋白酶／ＩｇＧＢＳＡ㊁诊断级ＢＳＡ共计８种，我们采取低内毒素ＢＳＡ（＜１ ０ＥＵ／ｍｇ），配合（１）㊁（２）措施，死亡率可降至２５％左右㊂（感谢上海中医药大学附属曙光医院肾病科；上海中医药大学中医肾病研究所高建东教授㊁史丽强住院医师的解答）０９中国比较医学杂志２０１８年１月第２８卷第１期㊀ＣｈｉｎＪＣｏｍｐＭｅｄ，Ｊａｎｕａｒｙ２０１８，Ｖｏｌ．２８．Ｎｏ．１。
慢性肾脏病血管钙化小鼠模型的建立
［ 关键 词］ 肾疾 病 ； 血管钙化 ； 疾病 模型 ， 动物 ［ 中 图分 类 号 ］ Ｒ ６ ９ ２ ． ９
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超抗原葡萄球菌肠毒素B诱导IgA肾病小鼠模型的建立
超抗原葡萄球菌肠毒素B诱导IgA肾病小鼠模型的建立史丽强;万强;吴燕升;刘伟伟;高建东【摘要】目的建立IgA肾病小鼠模型,并观察模型的生化及病理指标特点.方法 12只BALB/c小鼠随机分为正常组(6只)和模型组(6只),模型组单次尾静脉注射葡萄球菌肠毒素B(SEB)0.8 mg/kg,第二周开始,连续注射三周,第四周结束后检测小鼠24 h尿蛋白定量,尿微量白蛋白,肾功能BUN、Scr、UA;蛋白指标TP、ALB;肝功能ALT、AST、ALP;血脂TC、TG、LDL的情况,肾脏免疫荧光IgA的沉积,肾脏病理HE、PAS、PASM、Masson染色以及透射电镜的观察肾脏超微结构,以及肝脏与小肠HE染色、免疫荧光IgA的沉积变化.结果与正常组比较,模型组小鼠24 h尿蛋白定量与尿微量白蛋白均升高(P<0.01);模型组小鼠肾功能指标CREA,UA均高于正常组(P<0.05),BUN差异无显著性;模型组小鼠蛋白指标TP、ALB差异无显著性;模型组小鼠肝功能指标AST水平高于正常组(P<0.05),ALT、ALP差异无显著性;模型组小鼠血脂TG水平低于正常组(P<0.05),LDL水平高于正常组(P<0.01),TC差异无显著性.肾脏免疫荧光检查可见模型组小鼠肾小球系膜区呈颗粒状、团块状的IgA沉积;模型组小鼠肾脏病理轻中度损伤,系膜区免疫复合物增多;模型组小鼠肝脏HE染色可见少量炎性细胞浸润伴有部分肝细胞坏死,小肠绒毛缺损,肠绒毛变短变细、间距明显增宽,有部分分上皮脱落,中央央乳糜管扩张显著,淋巴细胞增多,明显可见炎性细胞浸润.结论使用超抗原SEB尾静脉注射小鼠可以成功复制IgA肾病动物模型.%Objective To establish a mouse model of IgA nephropathy and to observe its biochemical and pathological characteristics. Methods Twelve BALB/c mice were randomly divided into the normal group and model group, with 6 mice in each group. Mice in the model group received an intravenous injection of 0. 8 mg/kg superantigen staphylococcalenterotoxin B (SEB) into the tail vein once a week for three weeks. At the end of the 4th week, the mice were sacrificed, and the 24 h-urinary protein, urinary microalbumin, the renal function indicators BUN, Scr and UA were measured, levels of liver function indicators ALT, AST, ALP, and the blood lipid levels of TC, TG, and LDL were determined, the renal morphological changes were examined by pathology using HE, PAS, PASM and Masson staining, and by electron microscopy, the IgA deposition in the renal tissue was observed with immunofluorescence, and the liver and small intestine were observed by pathology using HE staining. Results Compared with the normal group, the mice of model group showed increased 24-hour urinary protein and urinary microalbumin (P<0. 01), increased CREA and UA (P<0.05), but not significantly changed BUN, TP and ALB. The liver function indicator AST was significantly increased (P<0. 05), but ALT and ALP were not significantly changed. The blood lipid TG was significantly decreased (P<0. 05) and LDL increased (P<0. 01), while the TC was not significantly changed. The kidney tissues had moderate histological changes, and immunofluorescence observation showed granular or massive IgA deposition in the renal glomerular mesangium. The liver tissue had some inflammatory cell infiltration and hepatocyte necrosis. The small intestine showed slender and shortened villi with widened inter-villous space and sloughed off epithelial cells, dilated central lacteal, and lymphocyte infiltration. Conclusions A mouse model of IgA nephropathy can be successfully established by tail vein injection of superantigen staphylococcal entrotoxin B.【期刊名称】《中国比较医学杂志》【年(卷),期】2017(027)012【总页数】7页(P102-108)【关键词】IgA肾病;动物模型;SEB;小鼠【作者】史丽强;万强;吴燕升;刘伟伟;高建东【作者单位】上海中医药大学附属曙光医院肾病科,上海中医药大学中医肾病研究所,肝肾疾病病证教育部重点实验室,上海市中医临床重点实验室,上海 201203;上海中医药大学附属曙光医院肾病科,上海中医药大学中医肾病研究所,肝肾疾病病证教育部重点实验室,上海市中医临床重点实验室,上海 201203;上海中医药大学附属曙光医院肾病科,上海中医药大学中医肾病研究所,肝肾疾病病证教育部重点实验室,上海市中医临床重点实验室,上海 201203;上海中医药大学附属曙光医院肾病科,上海中医药大学中医肾病研究所,肝肾疾病病证教育部重点实验室,上海市中医临床重点实验室,上海 201203;上海中医药大学附属曙光医院肾病科,上海中医药大学中医肾病研究所,肝肾疾病病证教育部重点实验室,上海市中医临床重点实验室,上海201203【正文语种】中文【中图分类】R-33IgA肾病(IgA nephropathy, IgAN) 是目前全球最常见的原发性肾小球疾病之一。
IgA肾病动物模型研究的新进展
IgA肾病动物模型研究的新进展
邱晓剑;陈扬;庄永泽
【期刊名称】《医学综述》
【年(卷),期】2006(12)5
【摘要】IgA肾病(IgAN)是最常见的原发性肾小球疾病,其发病机制目前仍不明确,为此人们设计或发现了许多IgAN动物模型,用以揭示、验证IgA肾病的发病机制.近年来IgAN的动物模型研究有了长足发展,出现了许多新的模型:子宫球蛋白缺陷模型、FcαRI(CD89)转基因小鼠模型、(New ZealandWhite× C57BL/6)F1-bcl-2转基因小鼠模型、HIGA小鼠模型、仙台病毒感染模型等.本文将就这些新进展进行综述.
【总页数】3页(P316-318)
【作者】邱晓剑;陈扬;庄永泽
【作者单位】福建医科大学福州总院临床医学院,福州,350025;福州市第二医院,福州,350007;福建医科大学福州总院临床医学院,福州,350025
【正文语种】中文
【中图分类】R6
【相关文献】
1.动物模型与IgA肾病的发病机制研究 [J], 王乾了;李贵森
2.IgA肾病动物模型研究的新进展 [J], 周金金;薛继强
3.IgA肾病动物模型的研究进展 [J], 金迪;张守琳;邹迪
4.IgA肾病动物模型研究现状 [J], 安丽丽;苏红;宋艳琦;郭登洲
5.IgA肾病肾小球硬化实验动物模型及其病理改变的研究 [J], 甄仲;黄文政;朱小棣;张威
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加速型肾毒血清肾炎模型在纯系小鼠的确立与泽泻在此肾炎模型的....ppt
腎毒血清腎炎是模擬人類因免疫引起所導致腎絲球腎炎的實驗模型，本研究藉投予兔子的抗基底膜 抗體血清而成功地誘發於純系小鼠上。臨床上治療腎臟疾病的漢方多用於改善浮腫、尿不利、煩渴 等，而澤瀉(Alismatis Rhizoma)是這類方劑主要組成之一；此生藥富含三萜類成分包括alisol A, B及 其相關成分，由於結構相似於類固醇，而可能有類似類固醇的藥理活性，故本研究評估澤瀉在此腎 炎模型的藥效。 誘發腎炎實驗以6週齡雌鼠(C57BL/6J mice)為宿主，先在小鼠足底皮下注射正常兔子之IgG；5日後 實驗組分三組分別尾靜脈注射腎毒血清NTS (100ml, 150ml, 200ml)，對照組則給予正常兔子血清。 藥效評估實驗依前述方法給予NTS 200ml，隔天起實驗組小鼠經口投予澤瀉濃縮劑Alisma orientale JUZEPCZUK, 澤瀉成分alisol B acetate, 類固醇 Methylprednisolone succinate (MPS), 與澤瀉併用MPS， 一天一次連續14天，而對照組則給予蒸餾水。小鼠每週一次使用特製新陳代謝器收集12小時尿液， 並在注射腎毒血清二週後用乙醚麻醉，全數犧牲以採集血清及腎臟組織。 測定尿蛋白含量與血清之尿素氮及肌胺酸酐值，來評估小鼠腎功能；其中之一枚腎組織使用PAS染 色觀察病理組織改變，另一則製成冷凍切片以進行免疫組織化學之卵白素-生物素複合物染色，選 擇一次抗體包括老鼠巨噬細胞F4/80, CD4+ 與CD8+ T細胞，及單核球趨化蛋白一(MCP-1)等，以辨 識發炎部位之特異性抗原。 實驗結果顯示，腎毒血清200ml實驗組之尿蛋白含量、血清之尿素氮及肌胺酸酐值皆明顯增加，且 14天內之組織觀察即可發現腎絲球肥大與腎微血管形成血栓而閉合、新月體的形成、腎間質細胞浸 潤、腎小管萎縮與輕微腎間質纖維化。在免疫組織化學染色中，腎組織有巨噬細胞、CD4+ T細胞浸 潤與MCP-1沈積呈色。此外，小鼠投予澤瀉濃縮劑4mg/kg, alisol B acetate, MPS, 與澤瀉併用MPS等 四組之尿蛋白、血清尿素氮及肌胺酸酐值顯著降低，鏡檢觀察皆有緩解病理組織惡化與減少細胞浸 潤，在此腎炎的療效依序是MPS, 澤瀉併用MPS, alisol B acetate, 與澤瀉4mg/kg。研究結果揭示腎毒 血清腎炎是因細胞性免疫而造成腎絲球損傷損傷，期望藉由阻斷此免疫路徑來作為未來治療腎絲球 腎炎之藥物研發方向。
肾小管坏死小鼠模型构建方法
肾小管坏死小鼠模型构建方法
【操作步骤】BALB/c小鼠，放置在每升空气中含有5mg的氯仿的环境中，持续3h后，可引起不同程度的肾坏死。
【结果分析】出生46～106d的雄性小鼠发生率可高达90％以上。
持续在氯仿气雾中24h，动物肾脏组织学观察，肾广泛损害且肾小管明显坏死。
7.8.2急性肾小管坏死大鼠模型
【操作步骤】SD或Wistar大鼠，体重190～250g，实验前置于代谢笼中饲养，记录食物、饮水和尿量。
实验时由尾静脉注射氯化汞1.0mg/kg。
24h后出现急性肾小管坏死表型。
【结果分析】尾静脉注射氯化汞24h后，尿量明显减少，尿渗透浓度降低，且出现细胞学的改变，近曲小管的末端部分可见肾坏死灶。
7.8.3硝酸钠、4-硝基儿茶酚等制剂引起的肾坏死模型
【操作步骤】成年雄性SD或Wistar大鼠，实验前置于代谢笼中饲养，测12h的尿量和尿中的酸性磷酸酶、碱性磷酸酶、乳酸脱氢酶、谷氨酰脱氢酶的正常排泄水平。
可选用下列不同药品给大鼠进行腹腔注射，造模。
(1)硝酸钠：2.5mg/(ml·100g)；
(2)4-硝基儿茶酚：1mg/(ml·100g)；
(3)4-硝基茶胂酸；2.5mg/(ml·100g)；
(4)4-氨基儿茶酚：1.5mg/(ml·100g)。
用药后12h测定尿量和尿酶量。
【结果分析】肾中毒的大鼠尿量普遍减少，乳酸脱氢酶、碱性磷酸酶排泄增多；酸性磷酸酶、谷氨酰脱氢酶排泄量在正常范围内。
肾组织表现广泛损害和肾小管明显坏死。
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IgA肾病小鼠模型的建立与鉴定【摘要】目的建立小鼠IgA肾病模型，为临床研究IgA肾病提供实验依据。
方法昆明种小鼠30只，分为正常对照组(10只)、模型组(20只)。
采用口服牛血清白蛋白(BSA)联合尾静脉注射葡萄球菌肠毒素B(SEB)的方法制作小鼠IgA肾病模型。
第12周末收集新鲜尿液，显微镜下尿红细胞定性检验;摘眼球取血，检测血肌酐(Scr)、尿素氮(BUN);光镜法观察肾小球系膜细胞及基质变化;电镜法检测系膜区有无电子致密物沉积;免疫荧光法观察系膜区IgA沉积情况。
结果第12周末，模型组小鼠均出现不同程度的血尿;Scr、BUN较正常对照组显著增高(P<0.01);光镜下系膜细胞及基质明显增生;电镜下系膜区电子致密物沉积;免疫荧光示系膜区大量的IgA沉积，而正常对照组则无上述表现。
结论 IgA肾病小鼠模型效果良好，病理、电镜及临床指标与人类IgA肾病临床病理相似。
【关键词】 IgA肾病;模型;血尿;免疫荧光;小鼠Abstract: Objective To establish mouse model of IgA nephropathy in order to provide experiment basis for the clinical research of IgA nephropathy. Methods 30 Kunming mice were randomly assigned into the control group (n=10) and the model group (n=20). Mouse model of IgA nephropathy was established by the method of oral intake of bovine serum albumin(BSA) together with injection of staphylococcus enterotoxin B (SEB) via the caudal vein. At the end of the 12 weeks, fresh urine was gathered for the quantitative test of hematuria; blood was taken through orbital sinus after eyeball extirpation to determine the serum creatinine (Scr) and blood urea nitrogen (BUN); the changes of mesangial cells and matrix in the kidneys were observed by light microscopy; electron microscopy was employed to detect electron-dense deposits in the mesangial area; the expression of IgA deposition was measured by immunofluorescence staining in the mesangial area. Results By the end of 12 weeks, there were different degrees of hematuria in model group mice. BUN and Scr levels in model group were significantly higher than in the normal control group (P<0.01). Light microscopy showed evident proliferation of mesangial cells and matrix. Electron microscopy demonstrated a high electron dense deposition in the mesangial area, while the above abnormalities were not observed in the normal control group.Conclusion The mouse model of IgA nephropathy proves effective, with pathological electron microscopic and clinical parameters similar to the clinical pathology of human IgA nephropathy.Key words: IgA nephropathy; model; hematuria; immunofluoresence staining; miceIgA肾病世界范围内广发的一种系膜增生型肾小球肾炎，占原发性肾小球疾病的10%～20%，中国、日本、新加坡等发病率较高，其中有20%～30%的患者进展到肾衰[1]。
建立与人类临床病理生理相似的IgA肾病动物模型，对研究IgA肾病的发病机理、临床治疗等具有重要的作用。
本实验采用口服牛血清白蛋白联合尾静脉注射葡萄球菌肠毒素的方法制作IgA肾病小鼠模型，为进一步探索IgA肾病提供实验依据。
1 材料和方法1.1 实验材料1.1.1 实验动物昆明种小鼠30只，鼠龄(10-12)周，体重20～26 g，雄性，动物编号SCXK(苏)2005-0005，由徐州医学院实验动物中心提供。
1.1.2 主要实验试剂和设备牛血清白蛋白(BSA,上海明睿生物有限公司，产品批号RF101-010);葡萄球菌肠毒素B(SEB,北京博迈世纪生物技术有限公司);兔抗小鼠IgA抗体(北京博奥森生物技术有限公司，抗体货号bs-0774R); 异硫氰酸荧光素(FITC)-标记的羊抗兔IgG(Millipore corporation); MicromHM340石蜡切片机;光学显微镜(日本Carton);日本Sysmex CA-1500自动生化分析仪;电镜(H-600)，切片机(LKBV型)。
1.2 实验方法1.2.1 酸化水的制备新鲜的双蒸水中加入盐酸，浓度为8.5 mmol/L。
即1 000 ml双蒸水中加入10%盐酸3.2 ml。
1%BSA酸化水的制备,1 g BSA加入100 ml酸化水。
1.2.2 造模方法适应性喂养1周后，取新鲜尿液进行显微镜下尿红细胞检测，均为阴性，将30只小鼠随机分为正常对照组(10只)、模型组(20只)。
正常对照组小鼠自由饮水与饮食;模型组小鼠除自由饮水和饮食外，前5周隔日予BSA(200 mg/kg)灌胃;第6周起予BSA(20 mg/kg，500 mg BSA溶于500 ml无菌生理盐水中)尾静脉注射，每天1次，连续3天;第8周起予SEB(0.5 mg/kg, 1 mg SEB溶于25 ml无菌生理盐水中)尾静脉注射，每周1次，连续3周，观察至第12周末。
1.2.3 IgA肾病小鼠模型的鉴定1.2.3.1 血、尿液检测第12周末留取小鼠新鲜尿液，行尿红细胞定性检测;经摘眼球取血(待测Scr，BUN)后，处死小鼠，留取右侧肾脏分别固定于10%甲醛及戊二醛中，行病理(光镜和电镜)及免疫荧光检查。
1.2.3.2 光镜检查小鼠肾组织经10%甲醛固定，石蜡包埋，5 μm切片,苏木精-伊红染色(H-E)，光镜下观察系膜区系膜细胞及基质有无增生。
1.2.3.3 电镜检查取材,前固定,漂洗,后固定,漂洗,脱水，置换及浸透，包埋及聚合，修块及切片，染色，电镜观察。
1.2.3.4 免疫荧光小鼠肾组织经10%甲醛固定，石蜡包埋，5 μm切片依次经二甲苯Ⅰ 20 min,二甲苯Ⅱ 20 min,95%乙醇 5 min,95%乙醇Ⅱ 5 min,双蒸水5 min×3次,3%H2O2 10 min,PBS 5 min ×3次,抗原修复(微波炉中高火5 min,低火15 min),自然冷却,PBS 5 min×3次,5%羊血清1 h,兔抗小鼠IgA(1∶100),4℃过夜,PBS 5 min ×3,FITC标记羊抗兔IgG(1∶100) 4℃过夜,PBS 5 min×3,甘油封片,荧光显微镜下观察摄像。
免疫荧光半定量标准:半定量标准参照目前国内通用的5级法(±～++++)分级:±：低倍镜下不能显示;+：低倍镜下似乎可见，高倍镜下可见;++:低倍镜下可见，高倍镜下清晰可见;+++：低倍镜下清晰可见，高倍镜下耀眼;++++: 高倍镜下刺眼。
1.3 统计学处理全部数据采用SPSS 13.0医学统计软件进行处理，计数资料用±s差表示，采用两独立样本t检验;等级资料用两独立样本非参数检验。
P<0.01为差异有统计学意义。
2 结果2.1 一般情况造模过程中，正常对照组小鼠无死亡，模型组小鼠先后死亡3只，分析原因可能是灌胃器误插入气管，或插入过深导致小鼠食管及胃黏膜损伤。
尾静脉注射SEB后，小鼠出现倦怠、少吃﹑少动等现象，2 h后恢复正常。
2.2 血、尿指标检测 12周末，采集新鲜尿液光镜下尿红细胞计数。
结果显示模型组小鼠明显高于正常对照组(P<0.01)(表1);摘眼球取血，留取血清，检测血Scr和BUN。
结果显示模型组较正常对照组明显升高(P<0.01)(表2)。
表1 2组小鼠尿红细胞计数的比较表2 2组血BUN和Scr的比与正常对照组比较：*P<0.012.3 镜下结构改变2.3.1 光镜模型组小鼠肾组织主要表现为肾小球毛细血管襻扩张，系膜细胞及基质明显增生，毛细血管腔部分闭锁，部分小管扩张;光镜下正常对照组小鼠肾小球，肾小管，及间质组织形态结构未见异常，系膜细胞及细胞基质未见增多(图1)。
图1 12周末，各组小鼠光镜下肾组织病理变化的比较(H-E染色，×400)A.模型组,肾小球系膜细胞及基质增生,毛细血管腔部分闭锁，部分小管扩张;B.正常对照组,肾小球系膜细胞及基质未见增生,毛细血管开放良好2.3.2 电镜模型组小鼠肾小球系膜区有大量电子致密物沉积，而正常组小鼠肾小球系膜区无或有少量的电子致密物沉积(图2)。
图2 12周末，各组小鼠电镜下肾组织病理变化的比较(×10 000)A.模型组,系膜区大量的电子致密物沉积;B.正常对照组,系膜区无电子致密物沉积2.4 免疫荧光检测 12周末，免疫荧光可以看到，系膜区IgA 荧光强度大多为+++～++++，而正常组系膜区IgA荧光强度为±～+，差异有统计学意义(表3、图3)。