作　　者： ; ; ; (高睿迪）;

机构地区： 南方医科大学

出　　处： 《生物技术通报》 2014年第3期187-192,共6页

摘　　要： 单纯疱疹病毒2型潜伏相关转录体(LAT)-RL1对放线菌素D诱导的凋亡作用的研究。构建重组pEGFP-RL1质粒,转染Vero细胞,RT-PCR及荧光鉴定重组质粒的表达。放线菌素D诱导Vero细胞凋亡,通过Hochest33342荧光染色观察细胞形态变化,流式细胞术检测细胞凋亡率,JC-1荧光观察膜电位变化,Caspase 3凋亡蛋白检测。RT-PCR和荧光观察表明该真核表达载体能在Vero细胞中高表达。Hochest33342染色转染了pEGFP-RL1的Vero细胞经放线菌素D凋亡诱导后,细胞形态正常。流式结果表明转染重组质粒pEGFP-RL1且经诱导凋亡组与正常对照组凋亡率无差异,而显著低于诱导凋亡组和转染空质粒pEGFP-C2诱导凋亡组。转染重组质粒pEGPF-RL1的细胞JC-1染色后,红色细胞的比例要远大于绿色细胞的比例,而转染空质粒pEGFP-C2被染成绿色细胞的数量较多。Caspase-3结果表明转染了空质粒pEGFP-C2的Vero细胞经诱导凋亡后,活性显著高于转染了pEGFP-RL1经放线菌素D诱导凋亡后的Vero细胞和正常Vero细胞。HSV-2 LAT RL1具有抗放线菌素D诱导的Vero细胞的凋亡作用。 It was to study the expression of herpes simplex virus type 2 latency-associated transcript（LAT）RL1 and its anti-apoptosis function induced by actinomycin D in vero cells. The recombinant plasmid pEGFP-ORF1 was constructed and transfected into Vero cells, expression was determined by green fluorescent protein and RT-PCR .The changes of Vero cells morphology induced by Actinomycin D were observed by Hochest33342 fluorescence staining, cells apoptosis rate was detected by flow cytometry . Membrane potential JC-1 fluorescence was observed, and Caspase 3 detected of apoptosis protein activity. Results showed that the green fluorescent protein has a high expressed efficiency in Vero cells, and target gene was detected by RT-PCR. Hochest33342 staining reavealed that Vero cells transfected with pEGFP-RL1 and induced apoptosis by actinomycin D had no changes in morphology. Flow cytometry assay showed that the cells apoptosis rate were no significant difference between pEGFP-RL1group and the normal group, but the cells apoptosis rate of pEGFP-RL1 was remarkable lower than the pEGFP-C2 group. Transfected with the recombinant plasmid pEGPF-RL1 cells by JC-1 staining, the proportion is much larger than red cells green cells transfected with empty vector pEGFP-C2. Caspase-3 results indicate that induction of apoptosis by transfected with empty vector pEGFP-C2 Vero cells, and the activity was significantly higher than those transfected with pEGFP-RL1 by actinomycin D-induced apoptosis of Vero cells and normal cells. HSV-2 LAT RL1 gene can effectively expressed in Vero cells and can protect Vero cells from apoptosis induced by actinomycin D.

关 键 词： 单纯疱疹病毒 放线菌素 凋亡 细胞

领　　域： [生物学]