Effects Of HBx Gene On Proliferation Of L02 Cells Under IL - 6 Intervention

Objective(1) To establish the cell model L02-HBx that stably expressed the protein, in order to do further research to find out the bionomics of HBx and the relationship between HBx and occurrence and development of hepatocellular carcinoma (HCC)(2) To observe the growth trends and cell cycles of L02-HBx expose to IL-6 and acquire their changes so as to stand the chance of breaking through liver regeneration and treatment of hepatic cirrhosis. We have been hoping that the experiments could supply the valuable data to direct the cytokine therapy for patients infected by HBV.Methods(1) To obtain the lowest concentration of puromycin that kill the L02 cytes by using the puromycin dosage-reaction test.(2) To establish the cell model L02-HBx that stably expressed HBx by Lentivirus transfection. We obtained the positive clones by the puromycin selection base on its lowest killing concentration.To culture the L02-HBx,L02-con and L02 respectively.After that, we detected the HBx DNA,mRNA and protein by PCR, RT-PCR and western blot respectively, which will assure that we establish the L02-HBx cell model successfully.(3) To culture the L02-HBx by the serum obtaining IL-6.At the same time To culture the L02-HBx without IL-6 synchronously as negative control. We observed the morphology of the L02-HBx, detected all groups about the proliferation with CCK8 arrays,in the meanwhile,we detected the cell cycles by FCM. Base on these results, We compared the L02-HBx with the other group about the proliferation and cell cycles.Results(1) Puromycin dosage-reaction test showed that the concentration of puromycin from 20 to 40μg/ml can kill all the cells,but the range from 0 to 10μg/ml can’t have the same effects,there also were several cells surviving,which determine that 20μg/ml was the lowest killing concentration.(2) We obtained the positive clones by puromycin selection in 5-7 days,the concentration for puromycin selection was 20μg/ml.To culture the L02-HBx,L02-con and L02 respectively.After that,the PCR, RT-PCR and western detection revealed that there were HBx DNA, HBx mRNA and HBx protein in the transcriptional level and HBx protein expression in the protein level respectively.(3) Inversion phase contrast microscope showed that the morphologic characteristic of L02-HBx cultured by the serum obtaining IL-6 had been changed more obviously than that of the L02-HBx without IL-6. The cells gather and pile up together.The figure become unregular and bigger.The borderline and the surface become obscure. The CCK8 arrays showed that L02-HBx proliferated more slowly than the control groups without exposing to IL-6. FCM showed that,in contrast with the control groups, the propotion of L02-HBx in S phase still falled but that in G2/M phase rose.Conclusions(1) The cell model L02-HBx that stably expressed HBx had been established successfully, which lay the foundation for us to study the bionomics of HBx and the relationship between HBx and occurrence and development of hepatocellular carcinoma (HCC)(2) L02-HBx proliferated more slowly when exposing to IL-6. And G2/M block appeared in cell cycle. After all, it possibly involves in the cell abberance and causes canceration. All the truth told us that the circumstances that the HBx was in would affect its biological functions.