【作者】 董娜；

【导师】 刘淑霞； 封晓娟；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2015， 硕士

【摘要】 目的:本研究以狼疮性肾炎模型小鼠MRL/1pr为研究对象,探讨狼疮鼠肾小球nestin和足细胞中活化型caspase-3的表达变化及其与小鼠蛋白尿的关系,以阐明足细胞损伤在狼疮性肾炎发病中的作用,为揭示狼疮性肾炎的发病机制及靶向治疗提供实验理论依据。方法:选取30周龄雌性MRL/MPJ小鼠和MRL/faslpr小鼠为研究对象,分别设为对照组和模型组。血、尿生化检测小鼠肾功能的改变;免疫组织化学技术和Western blot检测小鼠肾小球中活化型caspase-3蛋白表达情况;免疫组织化学技术检测小鼠肾小球中nestin蛋白表达情况;免疫荧光双染技术检测肾小球中活化型Caspase-3与Nestin共表达情况。结果:1.30周龄时狼疮性肾炎模型鼠尿蛋白水平升高与正常对照组相比,狼疮组小鼠24 h Upro明显升高,差异具有统计学意义(P<0.05),而BUN和Scr在两组之间比较无显著性差异。2.狼疮性肾炎模型小鼠肾小球上皮细胞出现足突融合:透射电镜结构显示,正常对照组小鼠肾小球基底膜及足细胞均无显著变化,模型组小鼠肾小球基底膜出现明显的不规则增厚,上皮细胞足突融合。3.活化型caspase-3在狼疮性肾炎模型鼠肾小球表达增高:免疫组织化学染色结果显示,活化型caspase-3阳性信号主要定位于细胞核和/细胞质,呈浅黄或棕色颗粒。在正常对照组小鼠肾小球几乎不见活化型caspase-3阳性表达,而模型组小鼠肾小球中可见活化型caspase-3蛋白表达升高;western blot结果显示,模型组小鼠肾小球中活化型caspase-3蛋白相对表达量显著高于正常对照组。4.狼疮性肾炎模型小鼠肾小球中nestin蛋白表达上调:免疫组化结果显示nestin阳性信号主要定位于细胞浆和细胞核,定量分析显示,与正常对照组相比,狼疮组肾小球中nestin蛋白表达上调。5.狼疮性肾炎模型组小鼠肾小球足细胞中活化型caspase-3表达升高:免疫荧光双染检测结果显示,在正常对照组只见nestin蛋白的阳性表达,未见明显nestin和caspase-3共定位表达,而在模型组小鼠肾小球中,nestin蛋白和caspase-3蛋白出现了明显共定位,呈黄色染色。6.相关性分析:狼疮鼠肾小球中活化型caspase 3蛋白的表达量与24h尿蛋白呈显著正相关(r=0.827,P=0.006)。结论:在狼疮性肾炎模型鼠肾小球足细胞中nestin蛋白及活化型caspase 3表达上调,且在肾小球中存在共定位,并与狼疮鼠蛋白尿水平呈显著正相关,提示足细胞骨架结构的改变和/或足细胞凋亡可能在狼疮性肾炎发病过程中发挥着重要的作用。更多还原

【Abstract】 Object: MRL/faslpr mice were used in this study to investigate the expression change of nestin and cleaved caspase-3 in mouse podocyte and its relationship with mice proteinuria level.Methods: Female MRL/MPJ and MRL/faslpr mice were respectively set as Control group and LN group, and urine protein and serum biochemical levels were measured to detect the mice kidney function; The expression of cleaved caspase-3 protein in mice glomeruli were detected by immunohistochemistry and western blot; in addition, the expression of nestin protein in mice glomeruli were detected by immunohistochemistry; And the co-localization level of nestin and cleaved caspase-3 protein were analysis by immunofluorescence.Results:1 The 24 h Upro level of 30-week female MRL/faslpr mice was increased. Compared with Control group, the 24 h Upro level of LN group mice was increased, and the difference was statistically significant; However, there was no significant difference in BUN and Scr level between two groups.2 The podocyte foot fusion was observed in mice glomeruli of LN group. The result of transmission electron microscopy showed that there was no significant structure change of glomeruli in Control group, and increased thickness of glomerular basement membrane and the podocyte foot fusion in the glomeruli were observed in LN group.3 The expression of cleaved caspase-3 protein was increased in glomeruli of LN group. Immunohistochemistry showed that, the positive signal of cleaved caspase-3 protein was shown as yellow granular, and mainly located in the nuclear and(or) cytoplasm of glomeruli cells. In Control group, no obviously positive signal was detected in mice glomeruli, and in LN group, the expression level of cleaved caspase-3 protein was obviously up-regulated. Western blot result showed that, the relative expression level of cleaved caspase-3 in mice glomeruli of LN group was significant increased then that in Control group.4 The expression of nestin was increased in glomeruli of LN group. Immunohistochemistry showed that, the positive signal of nestin protein was shown as yellow granular, and mainly located in the nuclear and cytoplasm of glomeruli cells. Compared with Control group, the expression level of nestin protein in mice glomeruli cells of LN group was significantly increased.5 The expression of cleaved caspase-3 in mice podocyte of LN group was increased. Immunofluorescence showed that, in glomeruli of Control group there was no obviously co-localization of nestin and cleaved caspase-3 protein, and in glomeruli of LN group significant yellow positive co-localization signal was detected.6 Correlation analysisi: In LN group, the 24 Upro level of mice was significantly positive correlated with the expression quantity of cleaved caspase-3 protein in glomeruli cells(r=0.827, P=0.006).Conclusions: the expression levels of cleaved caspase-3 and nestin protein in mice podocyte of LN group were both significantly up-regulated, and there was obviously co-localization of them, the 24 Upro level of mice was significantly positive correlated with the expression quantity of cleaved caspase-3 protein in glomeruli cells. All the results indicate that, the cytoskeleton change and apoptosis of podocyte maybe involved in the pathogensis of LN.更多还原