siRNA下调星形胶质细胞中PTEN表达对缺氧复氧诱导细胞凋亡影响

siRNA downregulation of PTEN expression in astrocytes on apoptosis induced by anoxia reoxygenation

目的研究siRNA下调星形胶质细胞中第10号染色体同源缺失性磷酸酶-张力蛋白(PTEN)表达对缺氧复氧诱导的细胞凋亡影响。方法分离培养乳鼠星形胶质细胞,用携带PTEN siRNA的慢病毒载体感染星形胶质细胞,经缺氧复氧处理后,用Real time PCR和Western blot检测细胞中PTEN表达变化。MTT测定细胞增殖活力,二硝基苯肼比色法检测乳酸脱氢酶(LDH)漏出率,流式细胞术测定周期和凋亡变化,Western blot检测细胞中细胞周期依赖性蛋白激酶4(Cdk4)、p21蛋白水平和胞质中细胞色素C(Cytochrome C)蛋白水平,JC-1法测定线粒体膜电位。结果携带PTEN siRNA的慢病毒载体感染后的星形胶质细胞中PTEN表达水平降低。缺氧复氧处理后的星形胶质细胞活力降低,细胞LDH漏出率升高,细胞凋亡率升高,细胞G1期比例升高,Cdk4蛋白水平降低,p21蛋白水平升高,胞质中Cytochrome C蛋白增多,线粒体膜电位降低。敲低PTEN可以提高缺氧复氧条件下星形胶质细胞活力,降低细胞LDH漏出率,减少G1期细胞比例,降低细胞凋亡率,促进细胞表达Cdk4蛋白,减少细胞表达p21,减少胞质中Cytochrome C蛋白,提高线粒体膜电位。结论敲低PTEN可以减少缺氧复氧诱导的星形胶质细胞凋亡,作用机制与线粒体凋亡途径有关。

Objective To study the effect of siRNA down regulating the expression of PTEN in astrocytes on apoptosis induced by hypoxia reoxygenation.Methods Astrocytes were isolated and cultured in milk rats,infection of astrocytes with lentiviral vectors carrying PTEN siRNA,after the anoxia and reoxygenation treatment,the expression of PTEN in cells was detected by Real time PCR and Western blot,MTT assay cell proliferation activity,the leakage rate of LDH was detected by two nitrophenyl hydrazine colorimetric assay,flow cytometry was used to determine the changes of cycle and apoptosis,Western blot was used to detect the level of Cdk4 and p21 protein and the level of Cytochrome C protein in the cytoplasm,the mitochondrial membrane potential was measured by JC-1.Results The expression level of PTEN in astrocytes infected with lentiviral vector carrying PTEN siRNA was decreased.After hypoxia reoxygenation,the viability of astrocytes decreased,the leakage rate of LDH was increased,the rate of apoptosis was increased,the proportion of G1 phase in cells increased,the level of Cdk4 protein elevated,the level of p21 protein reduced,the increase of Cytochrome C protein in the cytoplasm,the mitochondrial membrane potential decreased.Knocking down PTEN can increase the viability of astrocytes under anoxia and reoxygenation,decrease the leakage rate of cell LDH,decrease the cell proportion of G1 phase,decrease the rate of apoptosis,promote cell expression of Cdk4 protein,decrease the expression of p21 in cells,reduce Cytochrome C protein in cytoplasm,improve the mitochondrial membrane potential.Conclusion Knocking down PTEN can reduce the apoptosis of astrocytes induced by anoxia reoxygenation,the mechanism of action is related to the pathway of mitochondrial apoptosis.