沉默TRPV1基因对利多卡因所致细胞凋亡的影响与其机制.pdfVIP

摘要 亡增多。同时，沉默TRPV1基因可降低利多卡因的这些损伤作用。这提示TRPV1 与利多卡因神经毒性损伤机制有关。利多卡因可能通过激活TRPV1通道蛋白来 增加细胞内钙离子浓度，降低线粒体膜电位并介导细胞凋亡。 关键词：利多卡因；瞬态电压感受器阳离子通道；钙超载；凋亡 II 万方数据 Abstract ABSTRACT Objective： The aim is to construct a recombinant retroviral vector expressing shRNA targeting to human TRPV1 gene and observe its down-regulation effect in the U87-MG cell line.By silencing TRPV1gene,the effect on status and apoptosis was observed in U87-MG cell line after treated with lidocaine, thus to explore whether localanestheticproducingneurotoxicityby TRPV1. Methods: The oligonucleotides designed by Ambion on-line CAD software targeting to TRPV1were cloned into the pSUPERretro RNAi plasmid. The recombinant vector was confirmed by DNA sequencing and enzyme digestion analysis. The pSUPERretro-puro TRPV1 shRNA retrovirus was prepared by calcium phosphate method and transfected into U87-MG cells. After the screening by purinase, the expression levels of TRPV1 mRNA and protein were detected by fluorescent quantitation PCR and Western blotting. U87-MG cells were divided into control group, gene silencing group and empty vector group. For cells in each group, intracellular calcium ion concentration and mitochondrial membrane potential at different time point were study from the perspective of cell using flow cytometry after treated with lidocaine. Meanwhile, the cell vitality and cell apoptosis of U87-MGwasassayedby MTTand flow cytometry. Results: The expression vector pSUPERretro-puro TRPV1 shRNA was successfully constructed,which wasconfirmedby theDNA sequencing