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目的探讨醛糖还原酶(AR)对大鼠肾脏系膜细胞(MsC)表达细胞外基质成分纤连蛋白(FN)和Ⅳ型胶原(ColⅣ)蛋白的影响,以及AR在肾小球硬化病变过程中的可能作用及其机制。方法采用酶切、连接方法构建真核表达质粒pCDNA3-AR,采用脂质体介导以及G418筛选的方法,将pCDNA3-AR稳定转染至MsC中,应用逆转录聚合酶链反应(RT-PCR)、Western印迹法和免疫荧光检测转染MsC表达AR情况;以Western印迹法检测正常MsC、AR转基因MsC、应用AR抑制剂Sorbinil和Zopolrestat的正常MsC、转化生长因子-β1(TGF-β1)作用后的正常MsC的FN和ColⅣ蛋白表达情况。结果与正常MsC相比,TGFβ1作用后的正常MsCFN和ColⅣ蛋白表达水平分别增高1.6和1.7倍(P<0.01);AR转基因MsC的FN、ColⅣ表达分别增加1.8倍和1.5倍;应用Sorbinil时,FN和ColⅣ分别降低1.8倍和1.4倍(P<0.05);应用Zopolrestat时,FN和ColⅣ分别降低1.7倍和1.4倍(P<0.05)。结论AR高表达或活性受抑制时可以明显影响MsCFN、ColⅣ的蛋白表达,提示AR可能与肾小球硬化的病理过程有关。
Objective To investigate the effect of aldose reductase (AR) on the expression of extracellular matrix fibronectin (FN) and collagen Ⅳ (Col Ⅳ) in mesangial cells (MsC) of rats and the effect of AR on glomerular sclerosis In the possible role and mechanism. Methods The eukaryotic expression plasmid pCDNA3-AR was constructed by restriction enzyme digestion and ligation. The pCDNA3-AR was stably transfected into MsC by liposome-mediated and G418 screening. The expression of pCDNA3-AR was detected by reverse transcription-polymerase chain reaction ), Western blotting and immunofluorescence were used to detect the expression of AR in MsC. The normal MsC and AR gene MsC were detected by Western blotting. The normal MsC and TGF-β1 were treated with AR inhibitors Sorbinil and Zopolrestat After normal MsC FN and Col Ⅳ protein expression. Results Compared with normal MsC, the expressions of MsCFN and ColⅣ increased by 1.6 and 1.7 times (P <0.01), respectively. The expression of FN and ColⅣ in AR transgenic MsC increased by 1.8 times and 1.5 times respectively. When Sorbinil was used, FN and ColⅣ decreased by 1.8 times and 1.4 times respectively (P <0.05). When Zopolrestat was used, FN and ColⅣ decreased 1.7 times and 1.4 times respectively (P <0.05). Conclusions AR high expression or activity can be significantly inhibited when the expression of MsCFN, Col Ⅳ protein expression, suggesting that AR may be related to the pathological process of glomerular sclerosis.