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应用分子克隆技术将人FasLcDNA片段反向插入逆转录病毒载体pLXSN ,构建重组质粒pL (hFasL AS )SN ,转染包装细胞PA317后获得FasL反义RNA重组逆转录病毒表达载体假病毒上清 ,经NIH3T3细胞检测其感染滴度后转染肝癌细胞株HepG2细胞并筛选建系 ,命名为HepG2 hFasL AS。半定量RT PCR检测显示HepG2 FasL AS细胞FasL的mRNA明显少于正常HepG2细胞 ;FACS检测显示HepG2 FasL AS细胞FasL的表达与HepG2细胞相比显著下降 ;同时 ,HepG2 FasL AS细胞导致HL 6 0细胞凋亡能力有所下降。表明人FasL反义RNA重组逆转录病毒表达载体能抑制转染细胞FasL的表达并下调其致凋亡功能
The human FasL cDNA fragment was inserted into the retroviral vector pLXSN by molecular cloning technique to construct the recombinant plasmid pL (hFasL AS) SN, and the recombinant retrovirus expression vector pseudoviruses supernatant was obtained after transfection of packaging cells PA317. NIH3T3 cells were transfected into hepatoma cell line HepG2 after detecting the titer of the infected cells, and the selected strains were screened and identified as HepG2 hFasL AS. Semi-quantitative RT-PCR showed that FasL mRNA expression in HepG2 FasL AS cells was significantly less than that in normal HepG2 cells; FACS analysis showed that the expression of FasL in HepG2 FasL AS cells was significantly decreased compared with HepG2 cells; meanwhile, HepG2 FasL AS cells Death ability declined. The results showed that the recombinant retroviral vector expressing human FasL antisense RNA could inhibit the expression of FasL and down-regulate the apoptotic function of transfected cells