沉默ClC-3氯通道基因对PC-12细胞凋亡的影响
Effect of ClC？？3 chloride channel gene silence on apoptosis of PC？？12 cells

目的：观察氯离子通道蛋白3（ClC？？3）基因沉默对大鼠肾上腺嗜铬细胞瘤细胞PC？？12凋亡的影响。方法：取处于对数生长期的PC？？12细胞，随机分为4组：对照组（用正常培养基培养）、H2O2组（用300 nmol/L H2O2诱导12 h）、H2O2+空质粒组（细胞转染pGPU6/GFP 24 h后用H2O2诱导12 h）、H2O2+siRNA组（细胞转染pGPU6/GFP？？ClC？？3？？siRNA 24 h后用H2O2诱导12 h）。通过CCK？？8检测细胞存活率，流式细胞术检测细胞凋亡率，Western blot法检测Caspase？？3以及ClC？？3蛋白的表达，实时荧光定量PCR检测ClC？？3 mRNA的表达。结果：与对照组比较，H2O2组、H2O2+空质粒组和H2O2+siRNA组均表现为细胞存活率下降，细胞凋亡增加，ClC？？3 mRNA和Caspase？？3、ClC？？3蛋白表达增加（P<0.05）； H2O2组和H2O2+空质粒组比较，各指标差异无统计学意义（P>0.05）；但与H2O2组、H2O2+空质粒组比较，H2O2+siRNA组细胞存活率较高，细胞凋亡率较低，ClC？？3 mRNA和Caspase？？3、ClC？？3蛋白表达较弱（P<0.05）。结论：ClC？？3氯离子通道参与了H2O2诱导的PC？？12细胞凋亡。
Aim: To observe the effect of chloride channel protein 3(ClC？？3) gene silence on the apoptosis of rat adrenal pheochromocytoma PC？？12 cells. Methods: The siRNA sequences targeting ClC？？3 were synthesized. PC？？12 cells were allocated into 4 groups:control group, H2O2 group(cultured with 300 nmol/L H2O2 for 12 h), liposome group(pGPU6/GFP transfection and H2O2 culture) and siRNA group(pGPU6/GFP？？ClC？？3？？siRNA transfection and H2O2 culture).Cell survival was tested by CCK？？8 method, cell apoptosis was detected by flow cytometry,Caspase？？3 and ClC？？3 protein were detected by Western blot, and ClC？？3 mRNA was detected by Real？？time fluorescent quantitative PCR. Results: Compared with control group, cell survival rate decreased, while apoptotic rate, the ClC？？3 mRNA level, and the Caspase？？3 and ClC？？3 protein expression increased in the other 3 groups(P<0.05). The differences in the indexes mentioned above were not significant between H2O2 group and liposome group(P>0.05). Compared with those of the H2O2 group and liposome group, cell survival rate was higher, while apoptotic rate, the ClC？？3 mRNA level, and the Caspase？？3 and ClC？？3 protein expression were lower in siRNA group(P<0.05).Conclusion: ClC？？3 chloride channel might be involved in PC？？12 apoptosis induced by hydrogen peroxide