Study On Liver Transplantation Tolerance Induced By HLA-G

Organ transplantation is an effective or even only approach of treatment tosome end-stage human disease. Remarkable scientific progress towardunderstanding the biochemical basis necessary for transplantation has beenmade since the development of this technique at beginning of last century, buttissue rejection remains the primary clinical problem and challenge of organtransplantation. Early failure of the transplant no longer represents a significantthreat to graft loss since the introduction of the immunosuppressive agents willlikely diminish the problem of acute rejection and prolong survival oftransplanted organs. However, Immunosuppression would inevitably impairsystemic immunity, which in turn increases the risk of infections and cancer.Induction and maintenance of the donor-specific immune tolerance to transplantcould be the perfect status of transplantation and radically solve the problem ofrejection in clinical organ transplantation. It was reported that placentaltrophoblast cell surface nonclassical MHC-I molecules HLA-G protected targetcells from the cytotoxic activity of NK cells and T lymphocytes through direct orindirect interaction with several inhibitory receptor,suggesting that they may playan important role in maternal acceptance of fetal allograft. HLA-G expressionhas also been observed in thymic epithelial cells and tumor cells, which impliedthat the ectopic expression of HLA-G might be implicated in T cell tolerance anda way for tumor cells to escape from host immune surveillance. Therefore,HLA-G seem to be important molecules in inducing donor-specificimmunotolerance and its immunotolerance role bring promise for establishingimmunotelerance in organ transplantation. In the present study, we used RT-PCR to isolate the full length of HLA-G1cDNA and to analyze HLA-G1 expression in hepatocellular carcinoma tissue.The total RNAs prepared from tissue of human hepatocellular carcinoma were - 6 -博士学位论文 英文摘要used as templates, and the primers used were synthesized according to HLA-G1cDNA sequence. The RT-PCR result demonstrated that the mRNA level ofHLA-G1 was notably high in hepatocellular carcinoma tissue. Sequence analysisconfirmed that the insert is the full length of HLA-G1 cDNA sequence. To ourknowledge, this is the first evidence of the expression of the nonclassical HLA-Ggene in hepatocellular carcinoma tissue. We presume that the down-regulatedimmunosurveillance induced by highly expressed HLA-G1 may contribute to theoccurrence of HCC. To exam the biologic activities of HLA-G1, we subcloned HLA-G1 cDNA intopIRES2-EGF, and then transfected into MHC-I-cell line K562 cells usinglipofectamine reagents. The cells were subjected to selection of G418 (0.8mg/ml)and analyzed for HLA-G1 expression using Western blot. A resistant cell clonethat stably expressed HLA-G1, K562-G1, was used for analyzing the activity ofHLA-G1. LDH release assay was carried out to evaluate the protective effect ofHLA-G1 on cytolysis activity of NK cells. The experimental results showed that inthe E/T ratio of 80:1, 40:1, 20:1 and 10:1, the cytolysis activity of NK were 17.6%, 16.8%, 15.2% and 14.7% in K562-G1group, 91.0%,85.3%,45.3% and35.2% in K562 group and 89.3%, 84.2%, 36.5% and 32.1% in K562-emptygroup, respectively. The cytolysis activity of NK in K562-G1group is significantlylower than that in the control (P<0.01) . It suggested that the HLA-G1expressed in K562-G1 possess normal biological activities. It has been known that HLA-G has immunosuppressive activity in human andmice, but its function in rats remains unknown. To answer this question, we usedK562-G1 as target cells to investigate immunosuppressive effect of HLA-G1 onrat's NK and T cells. The results showed that in the E/T ratio of 80:1,40:1,20:1and 10:1, the cytolysis activity of NK were12.1%,11.6%,10.5% and 5.3% inK562-G1group, 23.5...