中文摘要：

目的观察HepG2细胞过表达FasL介导细胞凋亡的作用。方法从大鼠睾丸细胞中扩增出rFasL全长cDNA,亚克隆到T载体中,再克隆到pDC315载体中。转染HepG2细胞后用RT-PCR、Wester blot检测rFasL mRNA和蛋白表达,培养48h后收集细胞计数,进行比较分析。结果 rFasL cDNA序列与其在Genbank中的序列完全一致。rFasL真核表达载体转染HepG2细胞后能表达rFasL mRNA和蛋白;转染pDC315-rFasL的实验组HepG2细胞大量死亡。结论成功克隆了rFasL基因并构建其真核表达载体,证明能有效表达于HepG2中,表达FasL的HepG2细胞通过Fas-FasL结合介导周围及自身表达Fas的HepG2细胞凋亡。

英文摘要：

Objective To construct the eukaryotic expression vector and observe the effect of FasL gene transfection on induction of apoptosis of HepG2 cells.Methods RT-PCR and TA cloning technique were used to amplify the rFasL full length cDNA from rat testis cells,and then a eukaryotic expression vector pDC315 containing rFasL cDNA was constructed.Plasmid was transfected into HepG2 cells.The cells were harvested after 48h to identify the expression of plasmids by RT-PCR and Western blot.Meanwhile,two groups of HepG2 cells were respectively transfected with pDC315 plasmid as a plasmid control group and with H2O as a blank control group.After 48h of culture,the three groups of cells were counted,compared and analyzed.Results The sequence of cloned rFasL cDNA was consistent with that in GenBank.The HepG2 cells transfected with rFasL eukaryotic expression vector could express rFasL mRNA and protein.A large number of HepG2 cells died in the pDC315-rFasL experimental group.Conclusion The rFasL cDNA can be cloned and its eukaryotic expression vector can be constructed,indicating that rFasL gene can be expressed in HepG2 cells transfected with pDC315-rFasL.HepG2 cells expressing FasL induce apoptosis of neighbor-or auto-HepG2 cells,leading to their death.