基于siRNA技术的TRPC6基因沉默对血管紧张素Ⅱ诱导的足细胞自噬和凋亡的影响

Effects of silencing TRPC6 gene on the podocyte autophagy and apoptosis induced by AngⅡ based on siRNA technology

目的采用小分子干扰RNA(siRNA)干扰技术沉默足细胞相关分子瞬时受体电位阳离子通道蛋白6(TRPC6)对血管紧张素Ⅱ(AngⅡ)诱导的自噬和凋亡的影响。方法设计、构建siRNA,转染足细胞,使TRPC6基因沉默,采用流式细胞术、激光共聚焦、透射电镜及Western Blot技术检测不同浓度siRNA转染后TRPC6基因表达,优化获得TRPC6基因沉默最佳效果的条件。体外培养小鼠肾小球足细胞,设立对照组、AngⅡ组、空载体组、沉默TRPC6基因组和AngⅡ+沉默TRPC6基因组。对照组用含0.02%DMSO的RPMI 1640培养液培养;AngⅡ组加入AngⅡ(10^(-8)M)刺激足细胞;空载体组加入空载体至足细胞;沉默TRPC6基因组运用siRNA干扰技术沉默TRPC6基因;AngⅡ+沉默TRPC6基因组同时加入AngⅡ(10^(-8)M)及沉默TRPC6基因。处理12、24 h和48 h后分别收集细胞,采用流式细胞仪检测足细胞凋亡率,Western Blot检测TRPC6及自噬相关蛋白的表达,透射电镜及激光共聚焦电子显微镜观察自噬相关蛋白的分布。结果自噬在正常足细胞的表达量很微弱,AngⅡ组足细胞凋亡率明显升高,沉默TRPC6使足细胞凋亡率明显降低,与对照组比较差异有统计学意义(P<0.05)。透射电镜下AngⅡ组示足细胞超微结构发生改变,自噬表达增加,胞质中出现独立双层膜结构,胞质成分及溶酶体等细胞器形成双层及多层膜结构的自噬体。沉默TRPC6使自噬表达下降,与AngⅡ组比较差异有统计学意义(P<0.05)。激光共聚焦检测AngⅡ组LC3-Ⅱ的表达增加,沉默TRPC6使LC3-Ⅱ表达趋于稳定,与对照组比较差异有统计学意义(P<0.05)。结论AngⅡ促进足细胞的凋亡,沉默TRPC6基因能有效保护AngⅡ诱导的肾小球足细胞损伤,发挥保护足细胞的作用。

Objective To research the effect of silencing transient receptor potential cation channel protein 6(TRPC6) on autophagy and apoptosis induced by Ang Ⅱ.Methods The small molecule interfering RNA(siRNA) were designed to transfect podocyte and made TRPC6 genes silencing.Flow cytometry,laser confocal,transmission electron microscopy and Western Blot technique were used to detect the different concentrations of siRNA TRPC6 genes after transfection,and optimized for best effect of TRPC6 gene silencing.The mouse glomerular podocytes were cultured in vitro and were setted up the control group,AngⅡ group,empty carrier group,silenting TRPC6 gene group and AngⅡ + silenting TRPC6 gene group.The control group were cultured with 0.02% DMSO RPMI 1640 nutrient solution;AngⅡ group was joined Ang Ⅱ(10-8M) to stimulate podocyte;Empty carrier group was joined empty carrier to podocyte;In Silenting TRPC6 gene group,siRNA interference technology was used to silence TRPC6 gene;Ang Ⅱ +silenting TRPC6 gene group was joined AngⅡ(10-8M) and silenced TRPC6 gene at the same time.After12 h,24 h and 48 h,foot cells of the groups were collected respectively,and detected podocyte apoptosis rates using flow cytometry instrument.TRPC6 and autophagy related protein expression were detected with Western Blot.The autophagy related protein distribution were observated with transmission electron microscopy and laser confocal electron microscope.Results The autophagy expressing quantity was very small in the control group.the apoptosis rate increased significantly in AngⅡ group.Silenting TRPC6 made the podocye apoptosis rate significantly decreasing,and had statistical significance to compared with the control group(P < 0.05).Podocyte ultrastructure changed in the Ang Ⅱ group,autophagy expression increased,cytoplasm in independent double membrane structured,cytoplasmic components and lysosome organelles such as double and multilayer membrane structure of autophagy.Silenting TRPC6 decreased the expression of autophagy,and was statistically significant compared with Ang Ⅱ group(P < 0.05).Under laser confocal,LC3-Ⅱ expression increased in AngⅡ,silenting TRPC6 gene made the LC3-Ⅱ expression stable,compared with the control group,it was statistically significant(P < 0.01).Conclusion Ang Ⅱpromoted podocyte apoptosis,silenting TRPC6 gene can effectively protect podocyte injury which induced by AngⅡ,play the role of protecting.