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]
![一种IgA肾病小鼠肾功能不全模型的建立方法[发明专利]](https://img.taocdn.com/s3/m/7b7e98ea7e192279168884868762caaedd33bac8.png)
(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
ＩＡ肾病 （ｇ ｅｈｏａｈ ，ｇ 是具有 同一免疫病 理以 ｇ ＩＡ ｎｐ ｒｐｔｙ Ｉ ＡＮ）
Ｉ 为 主 的免 疫 球 蛋 白 在 。 球 系 膜 区 或 伴 有 毛 细 血 管 壁 的 在 一２ Ａ ｅ 肾小 ２℃切片机内用 ２ ５ ． ％聚乙烯醇冷冻包埋 ， 进行恒温冷冻
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。
THANKS
感谢观看
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。
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
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)。