Study On Tumor Formation Of Hepatocyte Transformed By HBV X Gene In Nude Mice And It's Mechanism

OBJECTIVE:In this study pCMVX/QSG7701 cell line expressing stably HBV X gene was used in order to understand the role HBV X gene in the pathogenesis of HCC.The main objectives are as follows:1.Try to investigate whether HBX alone is sufficient to directly transform the non-transformed immortalized human liver cell line QSG7701 and induce hepatocellular carcinoma in vivo.2.To make clear whether transplantation tumor is hepatocellular carcinoma or not and observe it's morphological changes.3.To explore roughly the pathogenesis of transplantation tumor in nude mice.METHODS:1.pCMVX/QSG7701 cells were vaccinated into subcutaneous tissue。of nude mice.pRcCMV2/QSG7701 and QSG7701 Cells were used as control.In 5 weeks after vaccination the nude mice were killed to observe whether a tumor is formed in nude mice.2.The activity state and food intake of the nude mice was recorded, and the texture,volume and metastasis of transplantation tumor were observed grossly.3.The transplantation tumor was observed microscopically on HE staining section.4.RT-PCR was used to detect the expression of mutant P53 and C-Myc mRNA in transplant tumor.5.Immunohistochemical SP method was used to analyze the protein expression of mutant P53 and C-Myc genes.RESULTS:1.The transplantation tumor occured within the subcutaneous tissue of the nude mice vaccinated with pCMVX/QSG7701 cells at the second week after vaccination.The tumor formation rate is 100%.No metastatic tumor was found in other organs.Transplant tumor was not formed in all negative control groups.2.The activity state,eating and drinking of the nude mice vaccinated with pCMVX/QSG7701 cells were normal,while their weights were increased gradually in the first 3 weeks.Since the 4^thweek,the nude mice's activity states were deteriorated and their weights were no longer increased.It's interesting that the mental state and eating of those nude mice inoculated with pRcCMV2/QSG7701and QSG7701 cells is normal, the weight is increasing all the time after inoculation.3.HE staining analysis confirmed that the character of transplant tumor was hepatocellular carcinoma.4.Mutant P53 mRNA and mutant P53 protein expressed in pCMVX/QSG7701,pRcCMV2/QSG7701,QSG7701 cells and transplantation tumor,the expression level of transplant tumor and pCMVX/QSG7701 cells was higher than that of pRcCMV2/QSG7701 and QSG7701 cells,respectively.The difference is significant(P＜0.01).5.C-Myc mRNA and C-Myc protein expressed in pCMVX/QSG7701,pRcCMV2/QSG7701,QSG7701 cells and transplantation tumor,the expression level of transplant tumor and pCMVX/QSG7701 cells was higher than that of pRcCMV2/QSG7701 and QSG7701 cells,respectively.The difference is significant(P＜0.01).CONCLUSION:HBX alone is sufficient to directly transform the non-transformed immortalized human liver cell line QSG7701 and induce hepatocellular carcinoma in vivo.HBx gene transform QSG7701 cells and induce hepatocellular carcinoma mignt by up-regulating the expression of mutant P53 and C-Myc genes.