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转染Megsin基因对糖尿病小鼠肾组织中P38MAPK、MCP-1和ICAM-1的影响

Effects of Megsin Gene Transfection on Expression of P38MAPK, MCP-1 and ICAM-1 in Renal Tissue of Diabetic Mice

【作者】 任广伟

【导师】 李英;

【作者基本信息】 河北医科大学 , 内科学, 2010, 硕士

【摘要】 目的:糖尿病(diabetes mellitus,DM)及其并发症已经成为一个公众的健康问题。糖尿病肾病(diabetic nephropathy, DN)是糖尿病最严重的并发症之一,是导致终末期肾衰竭(end-stage renal failure,ESRF)的主要原因。尽管我们对糖尿病肾病做了大量的研究,但肾脏损伤发生和发展的确切机制目前尚不清楚。如何早期有效的防治糖尿病肾病是目前国内外学者高度关注的课题。最近的研究报道糖尿病肾病的发生与众多的炎症因子和前炎症细胞因子的参与关系密切,如急性期反应蛋白、趋化因子和粘附分子等。Megsin是一种系膜细胞优势表达基因,定位于18q21.3,与丝氨酸蛋白酶结合,发挥丝氨酸蛋白酶抑制剂(serine protease inhibitor,serpin)的活性。Serpin的作用包括凝血、纤维蛋白溶解、炎症反应、细胞外基质代谢、细胞分化、增殖和凋亡等。推测megsin作为serpin的成员之一,可能参与了各种肾小球疾病中的多种紊乱进程。探讨megsin在糖尿病小鼠肾组织中的作用对于揭示糖尿病肾病的发病机制具有重要意义。本课题拟通过链脲佐菌素(streptozotocin,STZ)诱导建立糖尿病小鼠模型,并通过尾静脉质粒注射法,将megsin质粒、megsin-siRNA质粒及空质粒经由尾静脉注入糖尿病小鼠体内,以此分成三组,采用单侧肾切除组为正常对照组,观察不同组肾脏病变的程度,并采用免疫组化和western- blot方法检测不同组megsin、MCP-1、ICAM-1、磷酸化P38MAPK和P38之间的关系,研究megsin参与DN发生和发展的机制,可能为临床治疗糖尿病肾病提供新的干预途径。方法:选取清洁级健康雄性12周龄CD-1小鼠,共60只,行单侧肾切除,随机抽取45只腹腔注射STZ(溶于0.1mol/L柠檬酸缓冲液,浓度2%,pH值4.5,注射剂量150mg/kg)方法制备糖尿病小鼠模型,48小时后测定血糖、尿糖,血糖≥16.7mmol/L且尿糖持续阳性确定为糖尿病小鼠模型,随机将检测成模的45只糖尿病小鼠分为糖尿病+空质粒导入组(B组)、糖尿病+megsin质粒组(C组)和糖尿病+siRNAmegsin质粒组(D组),每组15只,将只做单侧肾切除的15只小鼠作为正常对照组(A组),采用尾静脉注射导入质粒载体,每周注射1次,整个实验期间小鼠自由饮食,不使用胰岛素及其它降糖药物。于实验开始后12周末收集血、尿及肾组织标本,检测血肌酐、24h尿蛋白定量及肾重/体重比值;石蜡切片行HE、PAS和MASSON染色观察肾小球病理改变,免疫组织化学染色和Western-bloting检测megsin、MCP-1、ICAM-1、磷酸化P38MAPK和P38表达情况,结果用均数±标准差(±s)表示,组间差异性比较采用单因素方差分析和q检验,显著性水平等于0.05,应用SPSS13.0软件包对所有数据进行统计学处理。结果:1 B组、C组、D组小鼠注射STZ 4~5天后即出现多饮、多食、多尿表现,三组无明显差异,而A组则无上述情况出现。12周末B组小鼠尿蛋白定量、血肌酐和肾重/体重比值升高(P<0.01),C组升高更为明显(P<0.01),D组尿蛋白和肾重/体重比值较B组降低(P<0.01)。2光镜HE显示2周和4周末B组肾小球内细胞数目较A组增多(P<0.01),C组增多更显著(P<0.01),12周末B组和C组小鼠肾小球内细胞数目减少,但仍高于A组(p<0.01),D组肾小球内细胞数目少于同期B组,有显著差异性(p<0.01);光镜HE、PAS、MASSON染色显示,第12周末B组肾小球体积增大,基底膜增厚,细胞外基质积聚,D组较B组病变减轻,C组病变最严重3免疫组化和western blot结果显示2周末B组Megsin、MCP-1、ICAM-1和磷酸化P38MAPK表达增强,C组变化更显著,D组较B组表达减弱,组间差异有显著性意义(p<0.01,p<0.05);4周末上述各指标变化更为明显,组间比较有显著性差异(p<0.01,p<0.05);12周末B组P38MAPK表达水平下降但仍高于A组和D组,弱于C组(p<0.01,p<0.05),其他指标如Megsin、MCP-1和ICAM-1表达进一步增强,C组增多更明显,D组表达较B减少(p<0.01,p<0.05)。结论:1 megsin在糖尿病小鼠肾组织中表达增强,可能与细胞外基质积聚、系膜细胞增殖、炎症细胞浸润、肾脏肥大及肾功能受损等相关,推测megsin在糖尿病肾病的发生发展中起一定作用。2过表达megsin糖尿病小鼠肾组织中MCP-1、ICAM-1和P38MAPK表达明显增强,提示megsin可能与上述因子发挥协同效应,促进了早期糖尿病肾损害的发生与发展,但其具体作用机制有待进一步研究阐明。3通过megsin siRNA质粒转染,megsin表达抑制后,MCP-1、ICAM-1和P38MAPK的表达减少,肾功能受损和肾组织病变减轻,显示抑制megsin的表达可以减轻糖尿病小鼠肾脏损害,为早期干预提供了一条新途径。

【Abstract】 Objective:Diabetes and its complications have become a public health problem. One of the most important complications is diabetic nephropathy, which is nowadays the main cause of end-stage renal failure. In spite of our greater understanding of this complication, the intimate mechanisms leading to the development and progression of renal injury are not well understood. How to effectively prevent and treat early diabetic nephropathy is currently a very hot subject concerned by scholars at home and abroad.Recent studies demonstrated that inflammatory cytokines and proinflammatory cytokines such as acute phase protein, chemokines and adhesion molecule probably play a major part in the development of diabetic nephropathy. Megsin is an mesangium-predominant gene,located in 18q21.3, binding with the serine protease,and play a serine protease inhibitor (serpin) activity. Serpin family is composed of a large number of members,their functions relate to blood coagulation, fibrinolysis, inflammation,cell proliferation, apoptosis,signal transduction, digestive and other systems. We speculate that megsin, as a member of the serpin, may participates in certain disfunction of various glomerular disease .Discussing megsin′s roles in renal tissue of diabetic mice has great significance to reveal the mechanism of diabetic nephropathy.We Constructed megsin siRNA expression plasmid and megsin cDNA expression plasmid,transfected them to STZ-induced diabetic mice, then investigate the relationship between megsin , phospho-p38MAPK,P38,MCP-1 and ICAM-1, to discuss the possible roles of megsin in inflammation of nephropathy, which may provide an important therapeutic targets that can be translated into clinical treatments for diabetic nephropathy. Methods:60 healthy male unilateral nephrectomy CD-1 mice were randomly divided to 4 groups.One of 4 groups was regarded as normal control group(A),then the others received a single intraperitoneal injection of STZ(dissolved in 0.1mol/L citrate buffer,pH 4.5)at a dose of 150mg/kg Wt.The diabetic model was considered to be successful when the blood glucose was 16.7mmol/L randomly and urinary glucose(+++)~(++++) after 72 hours of STZ injection.Then megsin plasmid,megsin-siRNA plasmid and control plasmid via the tail vein of mice were severally injected to diabetic control group(B),megsin plasmid group(C)and megsin-siRNA group(D).All mice were allowed free access to food and water during the experiment. At the end of 2 week,4 week,12 week,collecting the blood,24-hour urine and renal tissue samples,testing serum creatinine;24h urinary protein excretion rate,creatinine;kidney weight/body weight ratio; staining renal tissue by HE,MASSON and periodic acid - Schiff (PAS) , observed the changes in glomerular pathology in ordinary light microscope.The expression of megsin, phospho-p38MAPK,P38,MCP-1 and ICAM-1 in renal cortex was measured with immunohistochemical method and Western-Blot.The results were semiquantitatively analyzed with an image-processing system. All the data were analysed by SPSS13.0 statistics software,P value<0.05 was considered to have statistical significance.Results:1. The mice of group B,C and D all presented polydipsia, polyuria and polyphagia after 4 or 5 days of STZ injection.There is no significant difference in three groups.Kidney mass/body mass ratio,UP and Scr were higher in group B and C at 12 weeks(P<0.01),group C in those were highest(P<0.01),group D is less than group B(P<0.01).2.light microscopy:at 2 and 4 weeks,the number of glomerular cells in group B was more than in group A (p<0.01),but less than group C(p<0.01),the number of glomerular cells decreased in group B at 12 weeks ,but still higher than in group A (p<0.01), less than group C(p<0.01) ;glomerular hypertrophy, thickening of GBM and accumulation of ECM in group B were significantly higher than those in group A and group D,group C is the most serious.3. Immunohistochemistry and renal tissue protein Westen-bolt: The results of immunohistochemistry and Western blot showed that the expression of megsin, phospho-p38MAPK, MCP-1 and ICAM-1,at the end of 2 weeks, was obviously increased in group B and C than group A and D( (p<0.05,p<0.01). Group D is less than the same period of group B and C,there was a significant difference (p<0.05,p < 0.01). Changes of above-mentioned indicators were more significantly different among these groups at 4 weeks (p<0.05,p<0.01).At 12 weeks the expression level of phospho-p38MAPK decreased in group B,but still higher than in groupA and group D(p<0.05,p<0.01),other indicators’s expression such as megsin, MCP-1 and ICAM-1 further enhanced , there was significant difference (p<0.05,p<0.01).Conclusion:1 The expression of megsin enhanced in diabetic mice,that consisting with thickening of GBM,accumulation of ECM,inflammatory cell infiltration, mesangial cells hyperplasia,renal hypertrophy and renal dysfunction,suggesting that megsin may play a role in the development and progression of diabetic nephropathy.2 Over-expressed megsin may increase the expression levels of ICAM-1,MCP-1 and phospho-p38MAPK in renal tissue which may help explain the synergistic effect of megsin with other factors in promotes early diabetic nephropathy.However the specific ways have to be further studied.3 Megsin siRNA plasmid can decrease the expression level of megsin, ICAM-1,MCP-1 and phospho-p38MAPK,reduce 24-hr excretion of urinary protein,lower the level of creatinine,lessen the glomerular enlargement,mesangial cell proliferation and renal dysfunction that can be translated into clinical treatments for diabetic nephropathy.Megsin may be a new target for the therapy of diabetic nephropathy.

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