The Effect on the the Expression of p53、p21 and raf-1 in HepG2 Strain Stably Transfected by HBx Gene
|School||Central South University|
|Course||Clinical Laboratory Science|
|Keywords||Hepatitis B virus (HBV) HBx gene HCC apoptosis p53 pathway cell cycle raf-1 protein|
[ Objective] To fumble about constructing the laboratory condition of stable transfection of HBx gene and sieve the HepG2X cell model that can stably express X protein. To detect the biological parameter of the HepG2X cell strain that can stably express HBx gene and evaluate the reproductive ability of transfected strain. To study the possible carcinogenesis mechanism of HBx gene by signal transduction.[Methods] Eukaryotic expression vector containing HBx gene was transfected into HepG2 stain by liposome. HBx protein was detected by western blot. Cell growth curve, plate clone formatting experiment and MTS experiment were done to detect the reproductive ability of transfected cells. Cell apoptosis and cycle were detected by flow cytometer. p53 and raf-1 proteins were detected by western blot p21mRNA was detected by RT-PCR. In order to study the carcinogesis mechanism of HBx gene, the discrepancy was compared by the these indexes of HepG2X ,HepG2X0 and HepG2 strains.[Results] Eukaryotic expression vector containing HBx gene was introducted into HepG2 stain successfully. The strain that stably transfected by HBx gene was sieved. By western blot, X protein could be detect on the purposed location.The reproductive ability of transfected cells was higher than the control strains in the test of cell growth curve, plate clone formatting experiment and MTS test （P<0.01） .The results of flow cytometer cell apoptosis rate and cycle were abnormal in contrast of control stains. Compared by HepG2Xo and HepG2 strains, the expression levels of p53 and raf-1 proteins were increase in the strain of HepG2X . However, the level of p21 mRNA was decrease in this strain （P<0.01） . [Conclusions] The eukarytic transfected HBx gene HepG2 strain （HepG2X） was constructed and X protein could be detected in this strain. Heptitis B virus X protein can increase the apoptosis rate of the cells.p53-p21 apoptosis pathway is the possible way that the strain stably expressed HBx gene could increase the maglignant phenotype. HBx gene can increase the expression of the cell cycle protein raf-1.These results also cue that HBx gene induces HCC possibly by the derangement of cell cycle.