Nec-1对于环孢素A导致的细胞毒性的保护作用与机制研究

摘要

Objective To investigate the protective effect and its potential molecular mechanism of Nec-1 on cytotoxicity induced by cyclosporine A.Methods MRTEpiC, glomerular endothelial cell MGEC and mesangial cell line MMC were co-administered with Nec-1 and cyclosporin A in mouse renal tubular epithelial cell line, and then MTT assay and soft agar clone formation assay were used to detect Cell growth curve changes, clonal formation ability.Apoptosis was detected by flow cytometry.The expression of cyclin D1, CDK4, CDK2, Cyclin E and apoptosis-related Caspase 3 were detected by Western blot.Results After cyclosporine A action, the cell growth ability was significantly decreased and the clone formation ability was significantly decreased(P<0.05).Cyclin D1, CDK4, CDK2 and Cyclin E were significantly increased(P<0.05), but the ratio of apoptosis and the expression of Caspase 3 did not change.Nec-1 has obvious protective effect on cytotoxicity induced by cyclosporine A, which can increase the cell growth ability and clone formation ability, and reduce the cell cycle-related proteins Cyclin D1, CDK4, CDK2, Cyclin E.Conclusion Nec-1 has cytotoxic effect on the glomeruli and renal tubular cells by up-regulating the cell cycle-related proteins Cyclin D1, CDK4, CDK2 and Cyclin E, while Nec-1 has protective effect.%目的 探讨Nec-1对于环孢素A导致的细胞毒性的保护作用及其潜在的分子机制.方法 以Nec-1和环孢素A共同作用于小鼠肾小管上皮细胞株MRTEpiC、肾小球内皮细胞MGEC、肾小球系膜细胞株MMC,然后采用MTT法、软琼脂克隆形成实验分别检测细胞的生长曲线变化、克隆形成能力.采用流式细胞术检测细胞凋亡.采用Western blot检测细胞周期蛋白Cyclin D1、CDK4、CDK2、Cyclin E和细胞凋亡相关Caspase3.结果 环孢素A作用后,细胞生长能力明显下降,克隆形成能力明显降低,具有统计学意义(P＜0.05).细胞周期相关蛋白Cyclin D1、CDK4、CDK2、Cyclin E显著升高(P＜0.05),细胞凋亡的比例和Caspase 3的表达无变化.结论 Nec-1通过上调细胞周期相关蛋白Cyclin D1、CDK4、CDK2、Cyclin E对肾小球和肾小管细胞产生细胞毒性作用,而Nec-1具有保护作用.