Effect Of Ad-hgf Transfected Humen Trophoblastic Cells Anti Hbv Infection

Objective(1) To select an optional moltiplicity of infection(MOI) of recombinant adenovirus carrying hepotacyte growth factor( Ad-HGF) on JEGⅢcultured in vitro and detect the expression of HGF protein in Ad-HGF infected JEGⅢ, explore primarily the regulation effect of HGF on the JEGⅢand lay the foundation for the next experience.(2) JEGⅢwere infected with a recombinant adenovirus carrying hepotacyte growth factor(Ad-HGF) in an optional MOI and establish the cell model of Ad-HGF-JEGⅢinfected with HBV ,explore morphologically and quantitatively the effect of HGF on the Ad-HGF infected with HBV and provide the basis for the prevention from HBV intrauterine infection.Methods(1) JEGⅢwere infected with Ad-HGF at different MOI(10,25,50,100,200,400,800,1600),the Ad-GFP infected JEGⅢwere served as the control group. In order to select an optimal MOI, the degree of cell damage was assayed by MTT.The expression of HGF in JEGⅢtransfected with optional MOI Ad-HGF after 0,24,48,72h was measured with ELISA assay.(2) Based on the prophase study, JEGⅢwere cultured in 5%FBS and became starved. After spreading of 50%, JEGⅢwere infected with Ad-HGF at optimal MOI(200pfu/cell)after 48h, then the HBV positive serum(2×108HBV- DNA/L) was added up with a final concentration of 100DNA/ cell. After 24h of co-culture, the cells and supernatant were collected at 24,36,48,60,72h .The change of modality and structure of cells were observed with HE,GIMSA dyeing and transmission electron microscope, and the content of HBV-DNA was detected by fluorescent quantitation PCR.ALL experimental data were analyzed by SPSS 12.0 software.Results:(1) The optional MOI was selected as 200pfu/cel under the standard of high transfection efficiency and low cell damage.(2) After the transfection of Ad-HGF to JEGⅢat different times, the results of the expression of HGF detected by ELISA showed that Ad-HGF were transfected effectively into JEGⅢand the expression level of HGF presented a time-dependent manner in the primary 48h, and began to decrease at 72h.(3) The HE dyeing showed that theⅡgroup presented a obviously condens- ation of chromatin,marginalization,splitting of nuclear membrane and the apop- totic body than theⅠgroup, which is the typical modality apoptosis. TheⅠgroup and theⅡgroup have not shown the obvious changes of morphology. The results of GIMSA dyeing were similar to the one of HE dyeing.(4) The results of transmission electron microscope showed that the Ad-HGF transfected and HBV infected JEGⅢpresented the extension of mitochondria but without the round shape of HBsAg particle; the Ad-HGF untransfected and HBV infected JEGⅢpresented the cloudiness even disappear of mitochondria crest, the expanded endoplasmic reticulum with the widened perinuclear space and the expanded mitochondria with dim nuclear membrane, the round shape of HBsAg particle also observed in the cytoplasm, showing a vacuoles-shape;the dramatic increase of mitochodria were appeared in the Ad-HGF transfected group.(5) The content of HBV-DNA in the supernatant of Ad-HGF transfected JEGⅢat 24h,36h,48h,60h and 72h were lower than that of Ad-HGF untransfected JEGⅢ(P<0.05),which suggested that HGF has protection effect on trophocyte cells from the HBV infection.Conclusion:(1) HGF can be effectively transfected into JEGⅢand improve the proliferation of JEGⅢ.(2) HGF can efficiently suppress HBV infecting trophocyte cells.