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目的观察醛糖还原酶(AR)基因转染对体外培养大鼠肾脏系膜细胞(MsC)增殖的影响,并对其机制进行初步探讨。方法脂质体转染法将AR基因转入大鼠MsC,蛋白印迹鉴定转染效率。四甲基偶氮唑盐(MTT)比色法检测细胞增殖,流式细胞术分析细胞周期与凋亡,对比正常MsC和AR转染MsC的生长速度及AR抑制剂(ARI)Sorbinil和Zopolrestat对血小板源性生长因子-BB(PDGF-BB)和小牛血清(NBS)促MsC增殖作用的影响。蛋白印迹检测PDGF-BB作用后AR和c-Jun表达的变化,EMSA检测转录因子AP-1的活性。结果(1)转染MsCAR表达较正常MsC明显增高;(2)AR转染MsC较正常MsC生长速度快(P<0.01);(3)ARI可部分抑制PDGF-BB对正常MsC、AR转染MsC以及NBS对AR转染MsC的促增殖作用(P<0.01,P<0.05),而对NBS促正常MsC的增殖无显著影响;(4)PDGF-BB刺激MsC后可上调AR和c-Jun蛋白的表达,ARI可减弱c-Jun的这种表达增高;(5)PDGF-BB可活化转录因子AP-1,而ARI可削弱此作用。结论AR可能参与了MsC在病理情况下的过度增殖过程,其作用机制与AP-1活化有一定关系。
Objective To investigate the effect of aldose reductase (AR) gene transfection on the proliferation of rat mesangial cells (MsC) in vitro and its mechanism. Methods The gene of AR was transfected into rat MsC by lipofection method, and the transfection efficiency was identified by Western blotting. MTT assay was used to detect the cell proliferation. Cell cycle and apoptosis were analyzed by flow cytometry. The growth of MsC was compared with those of normal MsC and AR transfected with AR inhibitor (Sorbinil and Zopolrestat) Effects of platelet-derived growth factor-BB (PDGF-BB) and fetal bovine serum (NBS) on promoting the proliferation of MsC. Western blot was used to detect the changes of AR and c-Jun expression after PDGF-BB treatment. EMSA assayed the activity of transcription factor AP-1. Results (1) The expression of MsCAR in transfected MsCAR was significantly higher than that in normal MsC. (2) The growth of MsC in AR transfected with AR was faster than that in normal MsC (P <0.01). (3) ARI partially inhibited the expression of MsC and AR in PDGF- (P <0.01, P <0.05), but had no significant effect on the proliferation of normal MsC induced by NBS; (4) After PDGF-BB stimulated MsC, the up-regulation of AR and c-Jun ARI can decrease the expression of c-Jun. (5) PDGF-BB can activate the transcription factor AP-1, while ARI can weaken this effect. Conclusion AR may be involved in the process of MsC hyperproliferation in pathological conditions, and its mechanism may be related to the activation of AP-1.