Sig-1R基因表达抑制对内质网应激和足细胞损伤的影响
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国家自然科学基金(30872803、30971376);江苏省自然科学基金(BK2009046)


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

    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-

    Abstract:

    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.

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苏维维,丁桂霞,朱春华,袁杨刚,张爱华,黄松明. Sig-1R基因表达抑制对内质网应激和足细胞损伤的影响[J].南京医科大学学报(自然科学版),2011,(5):667-672

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  • 收稿日期:2010-11-05
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