Lentivirus Mediated HLA-G Gene Over Expression In Rat Primary Cultured Hepatocytes And Its Effect Of Anti-acute Rejection Of Rat Liver Transplantation

Objective:Human leukocyte antigen G (HLA-G) played a key role in maternal fetal immune tolerance, suggesting that it may be applied to organ transplant. The effect of inducing immune tolerance in vitro of HLA-G has been confirmed. The expression of HLA-G in transplant patients correlated with the decreased incidence of acute and chronic rejection. Grafts protective effect of HLA-G was proved only in rat skin transplant so far. We use HLA-G gene carried Lentivirus to transfect primary cultured rat hepatocytes and transplanted liver, with the purpose of investigating the anti-acute rejection effect of HLA-G in liver transplant in vitro and vivo.Method:1.260healthy male SD rats were used to practice rat orthotopic liver transplantation model in three stages. When operator was skilled, the other45SD rats and45Wistar rats were performed to establish SD to Wistar acute rejection model of rat liver transplantation.2. Construction of HLA-G carried Lentivirus system.3.The culture of primary hepatocytes from SD rat.4. Transfect SD rat’s primary hepatocytes with HLA-G carried Lentivirus, detect the fluorescence intensity, mRNA and protein of HLA-G through immunofluorescence, RT-PCR and Western-blot to evaluate transfection efficiency.5. We performed45SD to Wistar liver transplantation, they were randomly divided into three group. Experimental group was injected1×107TU/ml HLA-G gene carried Lentivirus into portal vein. FK506treated group was treated with FK506regularly after surgery. Control was treated with normal saline instead. HLA-G after surgery, AST, BIL of7,14,56days after surgery, biopsy and survival days were compared.Results:1. After surgical training, we successfully established SD to Wistar acute rejection model of liver transplantation.2. HLA-G carried Lentivirus could successfully transfected SD rat’s hepatocytes.3. Compared with the experimental group, FK506treated group (P<0.05) and control (P<0.01) got lower AST, BIL at7days. Experimental group had lower AST and BIL at12days and56days than control (P<0.01) and FK506treated group (P<0.01). The median survival time of control, FK506treated group and experimental group were12.00days,70.50days and91.50days (P<0.01), which suggested this kind of treatment could prolong recipients’survival time. The liver pathological results of experimental group and FK506treated group at7days showed mild rejection, however, in control showed severe rejection. After56days, the biopsy of FK506treated group showed the presence of mild rejection, experimental group was no rejection.Conclusion:1. As a therapeutic means for acute rejection model, HLA-G carried Lentivirus could effectively protect the function of rat liver graft and prolong survival days.2. Vitro experiments and experiments conducted in animal models indicated that hepatocytes were sensitive to Lentivirus mediated transfection and the transferred gene had long-term expression. Lentivirus had high transfection efficiency to hepatocytes, and it was promising in gene therapy of transplant related disease.