Sig-1R基因表达抑制对内质网应激和足细胞损伤的影响

Effect of knockdown of Sigma-1 receptor on endoplasmic reticulum stress and podocyte injury

目的:Sigma-1受体分子伴侣(Sigma-1 receptor chaperone,Sig-1R)是内质网中主要的伴侣蛋白,参与细胞凋亡等生物学行为,是多种疾病的治疗靶点。本研究旨在探讨Sig-1R基因表达抑制对内质网应激和足细胞损伤的影响,为Sig-1R及内质网应激在肾损伤中的作用提供新视点。方法:体外培养永生性小鼠足细胞系(MPC5),采用LipofectamineTM 2000与Sig-1R siRNA结合形成Sig-1R siRNA-脂质体复合物瞬时转染足细胞,设正常组、阴性对照组和Sig-1R干扰组。采用real-time PCR和Western blot检测Sig-1R的表达水平;Hochest染色法观察凋亡的足细胞核,计数凋亡率;采用Western blot检测足细胞nephrin、desmin、凋亡相关因子bcl-2、bax、caspase 3,8,9,12的表达水平。结果:①与对照组相比,干扰组Sig-1R核酸和蛋白表达均明显下降(P<0.05);②与对照组相比,干扰组足细胞裂孔隔膜蛋白nephrin表达显著下降,肌间蛋白desmin表达显著上升(P<0.05);③与对照组相比,干扰组中凋亡的足细胞数显著增加,抗凋亡蛋白Bcl-2表达明显下降,促凋亡蛋白bax表达明显增加,bcl-2/bax<1∶2(P<0.05),凋亡级联反应执行蛋白caspase-3明显活化(P<0.05);④干扰组中,内质网和线粒体途径相关凋亡蛋白caspase-12,9明显活化(P<0.05),而死亡受体相关凋亡蛋白caspase-8未见明显活化。结论:Sig-1R基因表达抑制可通过诱导内质网应激介导的足细胞凋亡,引起足细胞损伤,提示内质网应激部分参与Sig-1R诱导的足细胞损伤。

Objective：To explore the effect of knockdown of Sig-1R on endoplasmic reticulum stress and podocyte injury.Methods： siRNA vectors for silencing Sig-1R were constructed and transfected into mouse podocyte clones（MPC5）,which were divided into normal control,negative control and interference groups.The mRNA and protein expressions of Sig-1R were detected by real-time PCR and Western blot,respectively.The apoptosis of podocytes was detected by nuclear staining using Hochest 33258.The protein expressions of nephrin,desmin,bcl-2,bax,caspase-3,8,9,12 were detected by Western blot.Results：The reducation of Sig-1R in mRNA and protein levels was detected when the podocytes were transfected with Sig-1R siRNA（P 〈 0.05）.The expression of nephrin decreased,while the expression of desmin increased significantly（P 〈 0.05） compared to control.The apoptosis of podocyte was exhibited in the interference group.The expression of bcl-2 markedly decreased,while the expression of bax increased,and the ratio of bcl-2 to bax was less than 1/2.（P 〈 0.05）.The expression of active caspase-3 and caspase-12 increased significantly（P 〈 0.05）,while the expression of pro-caspase 9 decreased and pro caspase 8 had no change.Conclusion：The knockdown of Sig-1R can cause endoplasmic reticulum stress and podocyte injury.