Role Of Hepatic Stellate Cells In The Induction Of Tolerance Of Liver Transplantation In Rats

IntroductionNow the main problem of organ transplantation is: (1)although the short term survive rate of transplantation organ was very ideal, the rate of chronic rejection was also very high; (2)the long-term use of immunesuppression drug not only increased the fee and brought inconvenient of life, but also produced severe side effect. Since discovered the spontaneous immune tolerance in the animal model, transplantation specialist constantly viewed inducing organ and tissue immune torleance as a study aim. liver transplantation possess immune specificity, the reason is that (1) spontaneous immune tolerance often happen in the majority of experimental animal liver transplantation model; (2)the imunity of rejection in liver graft is often lower than other organ graft; (3)when simultaneously performed liver transplantation and other organ transplantation, the survive time of other organ will be prolonged; (4)about 25% recipient of liver transplantation can completely withdrawl immunesuppression drug. Liver was considered as a immune privilege organ, the important reason is that liver possess immune tolerance cells DCs(dendritic cells),KCs (kupffer cells),LSECs (Liver sinusoidal endothelial cells) and so on. However, whether HSCs(hepatic stellate cells) is a immune tolerance cell has not been definitioned. We try to reveal that HSCs is a immune tolerance cells and how the cells induce liver immune tolerance.Materials and Methods1. In vivo model of rat liver transplantation;2. Experimental groups: The rats were divided into two groups, Each had 5 pairs rats;(1) transplantation tolerance group: Lewis(donor) to DA(recipient);(2) transplantation rejection group: DA(donor) to Lewis(recipient);(3) the rats were sacrificed at the time of 7days;3. Transplantation rat HSCs were isolated and identified;4. Transplantation rat splenic T cells were isolated;5. Detected TLCs apoptosis after incubation with HSCs by Laser confocusing microscope and Flow Cytometry;6. Expression of FasL in HSCs surface by Flow Cytometry;7. Detected inhibition of T cells apoptosis after incubation with HSCs add with FasL antibody by Flow Cytometry.8. Detected expression change in tyrosine phosphorylation pathway protein of T cells after incubation with HSCs;9. RT-PCR for FasL, B7-H1 expression in HSCs and Fas expression in splenic T cells;10. Supernatant cytokine levels of IL-2,TNF-α, IL-10,TGF-β were determined by using ELISA;11. Pathology examination of transplanted liver;12. Expression of α -SMA in transplanted liver by immunohistochemistry;13. Statistical analysis: All values were expressed as mean±standard deviation of the mean (SD). t test of independent means were used for statistical analysis. Data were considered significant at a level of P<0.05.Results1. Apoptosis quantity of splenic T cells after incubation with HSCs in transplantation tolerance group was more than in transplantation rejection group;2. FasL expression in the surface of HSCs in transplantation tolerance group was more than in transplantation rejection group;3. FasL mRNA expression of HSCs and Fas mRNA expression of splenic T cells in transplantation tolerance group was more than in transplantation rejection group;4. FasL antibody could partial inhibit the role of HSCs inducing splenic T cells apoptosis;5. B7-H1 mRNA expression of HSCs in transplantation tolerance group was more than in transplantation rejection group;6. Th2 cytokine IL-10 and Th3 cytokine TGF- β expression of Supernatant in transplantation tolerance group was more than in transplantation rejection group;7. Tyrosine phosphorylation pathway 76KDa protein expression in transplantation tolerance group was more than in transplantation rejection group;8. HSCs infiltrated in liver of transplantation tolerance group was more than transplantation rejection group.ConclusionsInducing T cells apoptosis by HSCs isolated from transplantation tolerance group is via Fas/FasL and B7-H1 pathway, HSCs could increase Th2/Th3 cytokine secrete in transplantation tolerance group , the change of Tyrosine phosphorylation pathway 76KDa protein expression quantity result to HSCs inducing T cells apoptosis in transplantation tolerance group, then we can conclude that HSCs is a immune tolerance cell and may play a critical immunoregulatory role in peripheral specific immune tolerance in liver transplantation as DCs, KCs and LSECs.