| Construction of Recombinant Adenovirus Vector Carrying Human MDA-7/IL-24 and Its Expression on Hepatocellular Carcinoma Cell Line and Normal Liver Cell Line Objective To construct replication-incompetent adenovirus vector expressing MDA-7/IL-24 gene and to investigate its gene expressing in hepatocellular carcinoma cell lines and normal liver cell line in vitro.Methods Ad- EasyTM system was used to construct MDA-7/IL-24 by recombination in E.coli. The virus was packaged in 293 cells and subsequently identified valid. HCC (hepatocellular carcinoma) cell lines HepG2, SMMC7721, and Hep3B were infected with Ad.mda-7 and control virus with Ad.GFP respectively. The RNA expressing of MDA-7/IL-24 was indicated by PR-PCR and the protein expressing was demonstrated by ELISA assay.Results The construction of adenovirus vector expressing MDA-7 was successful, the expression of MDA-7/IL-24 were demonstrated by RT-PCR and ELISA assay in hepatocellular carcinoma cell lines HepG2, SMMC7721, Hep3B and normal liver cell line L02.Conclusion The construction of adenovirus vector expressing MDA-7 was successfully, and it should be a perfect vector for cancer gene therapy of MDA-7/IL-24. Adenovirus Vector Expressing MDA/IL-24, Melanoma Differentiation Associated Gene-7, Selectively Induces Growth Arrest, Apoptosis in Hepatocellular Carcinoma Cell Lines Objective To investigate the effect of MDA/IL-24 on the human hepatocullular carcinoma line HepG2, SMMC7721, Hep3B, MHCC97L, M6 and normal liver cell line L02 in vitro.Methods The MDA-7/IL-24 gene was transfected into human hepatocullular carcinoma cell line HepG2, SMMC7721, Hep3B, MHCC97L, M6 and normal liver cell line L02 with an replication-incompetent adenovirus vector. The mRNA expression of MDA7/IL-24 in HepG2, SMMC7721, Hep3B, MHCC97L, M6 and L02 cells was confirmed by RT-PCR. Protein expression was confirmed by ELISA assay. MTT assay and flow cytometry were used to study tumor cell proliferation and cell cycle in vitro. Hoechst and Annexin- V and PI staining were studied to indicate the apoptosis effect.Results It was confirmed by RT-PCR that the exogenous MDA-7/IL-24 gene expressed in HepG2, SMMC7721, Hep3B, MHCC97L, M6 and L02 cells. The protein product was confirmed by assay the supernatant with ELISA. MTT and apoptosis test indicated MDA-7/IL-24 can induce the human hepatocellular carcinoma cell HepG2, SMMC7721, Hep3B, MHCC97L and M6 growth suppression, apoptosis in vitro but not in normal liver cell line L02, cell cycle test revealed MDA-7/IL-24 can block cancer cell lines in G2/M but not in L02.Conclusion MDA-7/IL-24 selectively induces growth suppression, apoptosis in hepatocellular carcinoma lines HepG2, SMMC7721, Hep3B, MHCC97L and M6 in vitro regardless of the state of P53 gene but not in normal liver cell L02, means MDA-7/IL-24 can be a perfect gene for gene therapy in hepatocellular carcinoma(HCC). Study of Selectively Killing Mechanism of MDA-7/IL-24 in Hepatocellular Carcinoma CellObjective To investigate the selectively killing mechanism of MDA/IL-24 in hepatocellular carcinoma cell in vitro.Methods The MDA-7/IL-24 gene was transfected into human hepatocullular carcinoma cell line HepG2 and normal liver cell line L02 by replication-incompetent adenovims vector. The mRNA expression of MDA7/IL-24 and some apoptosis factor in HepG2 and L02 cells was confirmed by RT-PCR. Protein expression was confirmed by ELISA assay. Hoechst and Annexin-V and PI staining were studied to indicate the apoptosis effect. The relative protein of Bcl-2 family and Cytochrome C, Smac/Diablo and Caspase9 were detected with western blot.Results It was confirmed by RT-PCR, ELISA and western blot method that the exogenous MDA-7/IL-24 gene expressed in HepG2 and L02 cells, apoptosis test indicated MDA-7/IL-24 can induce the human hepatocellular carcinoma cell HepG2 apoptosis in vitro but not in normal liver cell line L02. It is confirmed that the expressing of protein which inhibited apoptosis Bcl-2 and Bcl-xL were increased in HepG2 after infected with Ad.mda-7, but not the same change in L02, at the same time, the Bax and Bak, facilitated the apoptosis process, were decreased in Bax, and the Bak had no change when infected with Ad.mda-7. Moreover, Ad.mda-7 infection facilitated the release cytochrome C and Smac/Diablo from mitochondria and then made the pro-caspase9 spilt to caspase9 and facilitating apoptosis.Conclusion MDA-7/IL-24 selectively induces growth suppression, apoptosis in hepatocellular carcinoma lines HepG2 in vitro but not in normal liver cell L02, and the mechanism is inhibiting Bcl-2 and Bcl-xL and increasing Bak, facilitated the cytochrome C and Smac/Diablo release from mitochondria and facilitated apoptosis with caspase9 eventually, means MDA-7/IL-24 can be a perfect gene for gene therapy in hepatocellular carcinoma(HCC).Treatment of hepatoma in nude mice by adenovirus-mediated mda-7/IL-24 combinedwith AdriamycinObjective: To investigate the anti-tumor effects of adenovirus-mediated mda-7/IL-24and/or Adriamycin on hepatocellular carcinoma (HCC) xenografted nude mice and toexplore a new way for hepatoma gene therapy combined with chemotherapy.Methods: The recombinant adenovirus vector carrying melanomadifferentiation-associated gene-7 (Ad.mda-7) was constructed; Ad.mda-7 and/or ADM wereinjected into the tumor-bearing mice. Their effects on the growth of the tumor and the survival rate of the mice were observed. The expression of mda-7 mRNA in tumor tissuewas analyzed with RT-PCR and western blot.Results: High level expression of mda-7 achieved in infected xenografted nude mousetumors with HCCLM6 by RT-PCR , ELISA and western blot. However, (1)in Ad.mda-7+ADM group, the hepatoma cell suppression percentage was 70.4%, while the percentage inADM group and Ad.mda-7 group were 35.9% and 46.3% (P<0.01) respectively; (2) InAd.mda-7+ADM group, the early death rate was 0, while the rate in ADM group was40%(P<0.01); in Ad.mda-7+ADM group, the survival percentage of nude mice was 60%,while the percentage in ADM group and Ad.mda-7 group were 0% and 40% respectively(P<0.05).Conclusion: Adenovirus-mediated human mda-7 gene can be expressed efficiently in vivo,Ad.mda-7 can significantly enhance the efficiency of ADM chemotherapy, and relieve thetoxic effects of ADM chemotherapy.
|
PDF Full Text Request