【作者】 蒋涛；

【导师】 张农； 郭慕依； 赵仲华；

【作者基本信息】 复旦大学 ， 病理学， 2005， 博士

【摘要】 肾小球硬化是临床多种肾小球疾病,如肾小球肾炎、IgA肾病、糖尿病肾病及狼疮性肾炎等导致慢性肾功能衰竭的重要病理基础,其主要病理特征是肾小球结构的毁损以及肾小球内细胞外基质(extracellular matrix,ECM)的异常积聚,而ECM合成和(或)降解失平衡被认为是导致肾小球硬化的重要发生机制之一。因此,近年来的研究热点已逐渐集中到ECM代谢的调控机制,及其与肾小球硬化发生、发展的相互关系上。 肾脏系膜细胞(mesangial cell,MsC)作为肾小球内功能活跃的固有细胞,不仅在维持肾小球正常结构和功能中具有重要地位,而且在急性肾小球炎症和以进行性肾小球硬化为特征的各种慢性肾小球肾炎疾病中也起着关键作用。体内外大量研究表明,肾脏MsC高表达转化生长因子β1(transforming growth factor β1,TGF-β1),可以通过直接促进ECM成分的合成或影响ECM合成和(或)降解主要调节酶系统的活性而促进ECM合成增加和沉积。因此,TGF-β1与肾小球硬化的发生发展有密切关系,是肾小球硬化病变发生发展过程中的重要介质之一。 我们课题组前期通过SSH-PCR研究发现,醛糖还原酶(aldose reductase,AR)是大鼠MsC中TGF-β1相关反应性基因之一。AR为NADPH依赖性醛-酮还原酶家族成员,是多元醇代谢通路中的限速酶。由于AR与糖代谢关系密切,所以以往对AR的研究主要是集中在糖尿病并发症中,例如糖尿病肾病、视网膜病等,并已有采用ARI进行糖尿病并发症实验治疗的研究报道。但新近的研究表明,AR发挥其某些作用、功能并不一定与高糖状态有关,其参与的很多病理过程也并无高血糖的诱因。因此,作为TGF-β1相关反应性基因之一,AR是否同样也参与了TGF-β1具有重要作用的、非高糖诱因所导致的。肾小球硬化等病理过程值得关注。 本课题拟采用制备大鼠抗Thy-1肾炎模型、细胞培养、基因转染、RT-PCR、Western blot以及EMSA等实验方法,分别从体内、体外两方面观察AR在肾小球肾炎、纤维化及硬化等病理过程中的表达及其与TGF-β1的相互关系;观察AR在TGF-β1诱导更多还原

【Abstract】 Progressive glomerulosclerosis, characterized by extracellular matrix (ECM) proteins accumulation and glomerular collapse, represents a common pathway of different renal diseases. There is increasing evidence that the imbalance of ECM synthesis and degradation may be an important mechanism of glomerulosclerosis. Recent research points are focused on the regulation ECM metabolism, and its correlation with the development of glomerulosclerosis.As inherent cells in glomerulus, the mesangial cells play an important role not only in maintaining the normal structure and functions of glomerulus, but also in acute glomerulonephritis and progressive glomerulosclerosis. Several reports have implicated transforming growth factor-pi (TGF-β1) in kidney diseases, including of glomerulosclerosis, since the MsC high expressing TGF-β1 can result in ECM deposition by inducing ECM proteins expression and/or changing the activity of MMPs/TIMPs.Our group had found AR gene was one of TGF-β1 -responsive genes by SSH-PCR. AR, one of the members of Aldo-keto superfamily, is the rate-limiting enzyme in polyol pathway, and several reports have implicated AR in diabetic mellitus, i.e. diabetic nephropathy, due to its functions in glucose metabolism. However, as one of TGF-β1-responsive genes, whether AR also has relations to glomerulosclerosis like TGF-β1 under normal glucose concentration remains unclear.In this study we used molecular biologic technologies, cell culture, and animal models to examine the expressin of TGF-pi and AR in glomerulosclerosis and the relations between them, and to observe the effect of AR in TGF-β -induced expression of ECM components, fibronectin and collagen IV, and to analyze the effect related signaling pathways and transcription factors. The purpose of our research was to elucidate the function and significance of AR in glomerulosclerosis, and provide new experiments data for enriching the pathologic mechanism of glomerulosclerosis.Part I The effect of TGF-pi on transcription and expression of AR in cultured rat MsCObjective To explore the effect of TGF-P 1 on transcription and expression of AR in cultured rat MsC.Methods Primary cultured rat MsC were treated with recombinant human cytokine TGF-pl in different concentrations or for different time. The transcription and expression of AR were examined by semiquantitative RT-PCR and Western blot analysis, respectively.Results The primary cultured rat MsC treated with TGF-p 1 showed increased transcription and expression of AR. When using the single concentration, 2ng/ml TGF-pl, the high transcription and expression were observed when incubated for 4h, and reached a peak at 12h. When using different concentrations for 12h incubation, the high transcription and expression were observed when used 0.5ng/ml TGF-pi, and reached a peak at lng/ml.Conclusions The recombinant human cytokine TGF-P 1 can up-regulate the transcription and expression of AR in dose and time-dependent manners in cultured rat MsC, which provide new proof for our former conclusion, namely AR gene was one of TGF-P 1-responsive genes.Part II The expression of TGF-p*l and AR in rat anti-Thy-1 glomerulonephritis model and human glomerulonephritis with different typesObjective To explore the expression of TGF-P 1 and AR in rat anti-Thy-1 glomerulonephritis model and human glomerulonephritis with different types and the relations between them.Methods Rabbit anti-rat Thy-1 serum were used to construct rat anti-Thy-1 glomerulonephritis model. RT-PCR, Western blot, immunohistochemistry and image analysis software were used to analyze the expression of TGF-P 1 and AR or the relations existing between them in rat anti Thy-1 glomerulonephritis model and human glomerulonephritis with different pathologic types.Results In rat anti Thy-1 glomerulonephritis model elevated expression level and AR were observed concomitant with the development of glomerulonephritis and the the correlation of them was positive by RT-PCR, Western blot, immunohistochemistry and image analysis. In human glomerulonephritis elevated expression level of TGF-PI and AR were also observed concomitant with the increasing severity of different types and the correlation of them was positive too.Conclusions AR indeed plays a role in glomerulonephritis or glomerulosclerosis based on these data: the increased expression of AR that was related with the development of rat anti-Thy-1 glomerulonephritis or severity of human glomerulonephritis were observed and the expression of AR related to the expression of TGF-pi. However the concrete role of AR in glomerulosclerosis remains unclear.Part III The effect of AR on TGF-pi-induced ECM components and the related signaling pathwayObjective To explore the effect of AR and AR on TGF-pi-induced expression of ECM components, FN and Col IV and the related signaling pathway or transcription factor.Methods Restriction endonucleases digestion and ligation were used to reconstructing eukaryotic expression plasmid pcDNA3-AR. Lipofectin was used to stably transfect AR vectors into MsC. RT-PCR, Western blot and immunofluorescence analysis were performed to verify the transfection. Western blot or EMSA were used to analyse the expression of fibronectin, collagen IV and MAPKs proteins or the activity of transcription factor AP-1 with or without the stimulation of TGF-|31.Results The normal MsC showed reduced expression of FN and Col IV after incubation with ARIs, and the MsC transfected with AR showed increased expression of FN and Col IV(P<0.05). After stimulation of TGF-pl, the normal MsC showed increased expression of FN and Col IV. The MsC preincubated with ARIs or transfected with AR showed obviously reduced or increased expression of FN and Col IV (TO.05). There was no change in expression of total MAPKs in different groups of MsC with or without stimulation of TGF-pi. The normal MsC showed increased expression of phospho-ERK, phospho-JNK,更多还原