CDNA Cloning, Expression Of Human Fas Ligand And Its Role In HepC₂ Cell Lines Apoptosis

Suppressing of apoptosis is thought as a mechanism of neoplasm occurrence, proliferation and metastasis. Fas(CD9S) transduces apoptotic signals upon cross-linking with the Fas ligand(CD95L), which is mainly existed in tumor-infiltrating lymphocyte (TIL) .To investigate Fas ligand and its physiological function in liver neoplasms,we evaluated the levels of soluble Fas ligand (sFasL) in a group of patients infected by Hepatitis B Virus (HBV)-induced chronic hepatitis, HBV-positive liver cirrhosis and hepatocellular carcinoma (HCC). Results show that FasL levels were significantly fell in patients with HCC when compared to the patients with hepatitis or liver cirrhosis.These findings emphasize the occurrence of circulating soluble FasL in patients with HCC, likely reflecting the involvement of neoplasm pathogenetic mechanisms.To further study, we constrcted recombinant eukaryotic expression vector pcDNA3.1hisB-FasL: According to the previously reported nucleic aid sequence of human FasL cDNA and characteristic of the expression vectors, two pairs of specific primers for FasL were designed and synthesized. The cDNA encoding human FasL was amplified from total RNA of human peripheral blood mononuclear cells (PBMC) by RT-PCR. Then the FasL cDNA fragments were cloned into pGEM-T Easy Vector to construct intermediate plasmids (PGEM-FasL) , at last the target fragments were subcloned into eukaryotic expression vector pcDNA3.1hisB . The correct recombinant pcDNA3.1hisB- FasL was selected by PCR and restriction endonuclease digestion and was confirmed by DNA sequencing respectively?Then, the recombinant pcONA3.1hisB- FasL was tranfected into human hepatoma cell line HepG2 by lipofection.After cultured 24 hours,we found FasL expressed in HepG2 cytoplasm and surface, ,sFasL can also be accumulated insupernatants.These means FasL was transfected into the cell and began to be expressed. These transfected HepG2 cell were co-cultured with HepG2.2.15 (HepG2 transfected with HBV DNA,by which, Fas can be fully expressed.) Subsequently a significant proportion of cells became apoptotic, as evidenced by positive staining of annexin V and propidium iodine.Therefore, this study supports a novel pathway of hepatoma cells were apoptotic via the Fas-Fas ligand system induced by itself,but not tumor-infiltrating lymphocyte (TIL).