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目的研究RNA干扰(RNAi)技术能否有效抑制三磷酸甘油醛脱氢酶(GAPDH)的表达以及对鱼藤酮所致神经毒性的影响。方法体外化学合成针对GAPDH基因的小干扰RNA(siRNA),通过脂质体Lipofectamine 2000瞬时转染后采用实时荧光定量PCR(real-time PCR)和蛋白免疫印迹分析(Western blot)进行干扰效果的鉴定;使用激光共聚焦荧光显微镜观察干扰对鱼藤酮诱导的GAPDH定位和结构改变的影响;用流式细胞仪检测线粒体膜电位变化。结果实时荧光定量PCR及蛋白免疫印迹分析显示,化学合成的siRNA可有效抑制细胞内GAPDH mRNA和蛋白水平的表达;激光共聚焦荧光显微镜观察显示,干扰GAPDH表达的细胞可减少鱼藤酮所致的GAPDH的聚集及核转位;流式细胞仪检测发现干扰GAPDH的表达可部分抵抗鱼藤酮所致的细胞线粒体膜电位的下降。结论下调GAPDH的表达可以减少鱼藤酮所致的细胞内GAPDH的聚集和核转位,减少线粒体膜电位的下降,提示GAPDH可能是鱼藤酮所致神经毒性的重要分子靶点。
Objective To investigate whether RNA interference (RNAi) can effectively inhibit the expression of glyceraldehyde triphosphate dehydrogenase (GAPDH) and the neurotoxicity induced by rotenone. Methods Small interfering RNA (siRNA) against GAPDH gene was synthesized in vitro and transiently transfected by Lipofectamine 2000. The real-time PCR and Western blot were used to identify the interference effect The effect of interference on rotenone-induced GAPDH localization and structure change was observed by laser confocal fluorescence microscopy. The change of mitochondrial membrane potential was detected by flow cytometry. Results Real-time quantitative PCR and Western blotting analysis showed that chemically synthesized siRNA could effectively inhibit the expression of GAPDH mRNA and protein in cells. Confocal laser scanning microscopy revealed that GAPDH-induced GAPDH Aggregation and nuclear translocation; flow cytometry showed that interference with the expression of GAPDH could partly resist the decline of mitochondrial membrane potential induced by rotenone. Conclusion Down-regulation of GAPDH can reduce the intracellular GAPDH accumulation and nuclear translocation induced by rotenone and decrease the mitochondrial membrane potential, suggesting that GAPDH may be an important molecular target of neurotoxicity induced by rotenone.