RNA干扰靶向抑制三磷酸甘油醛脱氢酶表达对鱼藤酮所致神经毒性的影响

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Lipofectamine2000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。

The effect of targeting inhibition of GAPDH expression by RNA interference on rotenone induced cell death

Objective To investigate the effect of GAPDH siRNA on rotenone induced cell apoptosis. Methods The small interfering RNA （siRNA） specific targeted to GAPDH gene was constructed by chemical synthesis. SiRNA was transfected into nerve growth factor differentiated PC12 cells and identified by real-time PCR and western blot analysis. Laser scanning confocal microscope was used to study the location and aggregation of GAPDH. The mitochondria membrane potential changes of transfected or unvalidated negative siRNA transfected cells after rotenone exposure were detected by rhodamine 123 staining using flow cytometry. Results Real time PCR and western blot analysis showed that GAPDH siRNA can efficiently suppress GAPDH expression. Laser scanning confocal microscope found that GAPDH become aggregated in the cytosol and nuclear translocation in rotenone treated PC12 cells;while GAPDH siRNA treated cells has lesser GAPDH aggregation and nulcear translocation. As detected by flow cytometry, down-regulation of GAPDH significantly decrease rotenone induced mitochondriai membrane potential collapse, compared with unvalidated negative control. Conclusions Down-regulation of GAPDH suppresses rotenone induced GAPDH aggregation and nuclear translocation, decrease rotenone induced mitochondrial membrane potential collapse and cells apoptosis, which may play a role in rotenone induced dopaminergic neuron death.