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目的探讨肿瘤坏死因子α(TNFα)对人肾小球系膜细胞(HMCs)IP3R1蛋白和mRNA表达的影响及PKC在此信号通路中的作用,以期为肝肾综合征(HRS)肾小球滤过虑下降的发生机制和防治思路提供理论依据。方法用实时定量PCR和免疫印记法检测IP3Rl mRNA和蛋白表达的情况,并分别用PMA耗竭内源性蛋白激酶C(PKC),PKCα特异性抑制剂Safingol和PKCδ特异性抑制剂Rottlerin及HA-DN-PKCα质粒转染干预上述诱导实验。同时,用免疫印记法检测TNFα对PKCα和p-PKCα表达的影响。用非放射性测活检测TNFα对PKCα的活化及抑制剂对PKCα活化的影响。结果 TNFα作用HMCs后,IP3R1蛋白和mRNA表达均明显增加。PMA,PKCα抑制剂Safin-gol,HA-DN-PKCα质粒转染均能明显阻断TNFα诱导的IP3R1蛋白的上调,而PKCδ抑制剂Rottlerin无明显阻断作用。TNFα刺激HMCs,各不同时间组总PKCα蛋白表达无明显差异,而TNFα刺激8h p-PKCα蛋白表达明显增加。此外,非放射性测活表明TNFα活化PKCα,Safingol可阻断PKCα的活化。结论 TNFα能上调HMCs中IP3R1蛋白及IP3RlmRNA的表达。活化的PKCα在TNFα上调IP3Rl的表达中起重要作用。
Objective To investigate the effect of tumor necrosis factor α (TNFα) on the expression of IP3R1 protein and mRNA in human glomerular mesangial cells (HMCs) and the role of PKC in this pathway. To consider the mechanism of decline and prevention and control ideas to provide a theoretical basis. Methods The expression of IP3R1 mRNA and protein were detected by real-time PCR and Western blotting. The depletion of endogenous protein kinase C (PKC), PKCα specific inhibitor Safingol and PKCδ specific inhibitor Rottlerin and HA-DN -PKCα plasmid transfection intervention induction experiments. Meanwhile, the effect of TNFα on the expression of PKCα and p-PKCα was detected by Western blotting. The activation of PKCα by TNFα and the effect of inhibitor on the activation of PKCα were detected by non-radioactive assay. Results After treated with TNFα, the expression of IP3R1 protein and mRNA were significantly increased. PMA and PKCα inhibitors Safin-gol and HA-DN-PKCα plasmid could obviously block the TNFα-induced upregulation of IP3R1 protein, while PKCδ inhibitor Rottlerin had no obvious blocking effect. TNFα stimulated HMCs, there was no significant difference in total PKCα protein expression between different time groups, while TNFα stimulation 8h p-PKCα protein expression increased significantly. In addition, non-radioactive assays indicate that TNFα activates PKCα and that Safingol blocks PKCα activation. Conclusion TNFα can up-regulate the expression of IP3R1 protein and IP3R1 mRNA in HMCs. Activated PKCα plays an important role in the up-regulation of IP3R1 expression by TNFα.