反义RNA阻断Hep G2细胞FasL的表达及功能研究

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

Construction and Functional Determination of Human FasL Anti-Sense RNA Recombinant Retrovirus Vector

FasL(CD95 ligand) is an important cell surface molecule which involves in many physiological and pathological procession Here, we inserted human FasL cDNA reversely into recombinant retrovirus vector plasmid pLXSN using subclone approach This recombinant plasmid was transfected into retrovirus packaging cell line PA 317 and the harvested culture supernatant was followed by determination of titer of the recombinant retrovirus, which could transcript anti sense RNA of hFasL Human hepatoma cell line Hep G2, expressing FasL in routine environments, was transfected with the retrovirus and screened Semi quantative RT PCR and FACS were carried out to test variance of the FasL expression level before and after being infected with the retroviral vector In addition, difference of apoptosis inducing capacity of Hep G2 was tested by co incubating with Fas positive HL 60 at different effect target ratio Our data showed that FasL expression level and apoptosis inducing capacity of Hep G2 was down regulated significently by the antisense RNA retroviral vector