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目的:构建FLIP(FLICE-inhibitory protein)腺病毒表达载体并观察该载体在大鼠肺血管内皮细胞(PVEC)中的表达情况,为研究FLIP蛋白的抗凋亡作用打下基础.方法:利用含有大鼠FLIP基因的原始载体,通过Admax腺病毒包装系统,构建表达大鼠FLIP基因的重组腺病毒Ad-FLIP;原代培养并鉴定大鼠PVEC.利用成功构建的重组腺病毒Ad-FLIP感染大鼠PVEC.通过逆转录酶-多聚酶链反应(reverse tran-scriptase-polymerase chain reaction,RT-PCR)和Western Blot分别检测经重组腺病毒Ad-FLIP感染后大鼠PVEC及对照组细胞中FLIP基因mRNA和蛋白表达水平.结果:成功构建含有大鼠FLIP基因的重组腺病毒Ad-FLIP;经Ⅷ因子间接免疫荧光鉴定原代培养大鼠PVEC纯度达90%以上;大鼠PVEC经重组腺病毒Ad-FLIP感染后24 h即检测到FLIP mRNA和蛋白表达水平明显增高.结论:重组腺病毒Ad-FLIP可有效提高大鼠PVEC中FLIP基因的表达水平.
OBJECTIVE: To construct a FLIP (FLICE-inhibitory protein) adenovirus expression vector and observe the expression of the vector in rat pulmonary vascular endothelial cells (PVEC), so as to lay the foundation for studying the anti-apoptotic effect of FLIP protein.Methods: Mouse FLIP gene was used to construct recombinant adenovirus Ad-FLIP expressing rat FLIP gene by Admax adenovirus packaging system.PVEC was cultured and identified in primary culture.The recombinant adenovirus Ad-FLIP was used to infect rat PVEC.The mRNA and protein levels of FLIP in PVEC and control cells were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western Blot respectively after infection with recombinant adenovirus Ad-FLIP The recombinant adenovirus Ad-FLIP containing rat FLIP gene was successfully constructed.Immunofluorescence assay with indirect immunofluorescence assay showed that the purity of PVEC in primary cultured rat was over 90% The expression of FLIP mRNA and protein was detected at 24 h after infection.Conclusion: Recombinant adenovirus Ad-FLIP can effectively improve the expression of FLIP gene in PVEC.