| ObjectivesThe objectives of the study were to explore the role of blocking B7/CD28 costimulation pathway with CTLA4-Ig gene in inducing tolerance after liver transplantation. Its relative mechanism was discussed too. Rat orthotopic liver allograft model was employed in this study. CTLA4-Ig gene was transducted into allograft with aid of adenovirus encoding P -lactase and serving as control in this study. The effect of recombinated CTLA4-Ig fussion protein on the function of macrophages was also studied in vitro in this research.Methods1. Establishing orthotopic liver transplantation model using SD rats to Wistar rats.2. Grouping: Group I (syngeneic control): Wistar to Wistar; GroupII (rejection control): SD to Wistar; Group IIKCsA-treated group): SD to Wistar recipients were treated with Cyclosporine A 3. Omg. kg"', d"1 intramuscllarly after allografting from day 0 to day 12 postopration. Group IV (CTLA4-Ig-transducted group): SD to Wistar, the donor was pretreated with CTLA4-Ig-transducted recombinated adenovirus at dosage of 1X 109PFU, once 7 day before transplantation; Group V(LacZ+control):SD to Wistar, the donor was pretreated with LacZ+ gene-transducted adenovirus, by method as fore mentioned.3. Parameters: general situation and survival of each group were observed on 1,3,5,7, 12d. 3 rats were sacrificed. Blood samples were harvested for detecting of IL-2, IL-4, IL-10 and IFN-y by ELISA. Expression of cytokine in grafts was measured by reverse-transcription polymerase chain reaction. Respectively, the expression of CTLA4-Ig gene and the infiltrate of macrophage and CD8T cell in grafts were detected by immunolhistochemistry stainning. Cell apoptosis in grafts was detected by TUNEL. Morphologic changes of graft and severity of rejection were assessed too.4. Studies in vitro: the macrophages were isolated from peripheral blood of volunteers and stimulated with or without lOng/ml LPS for 6 hours, then the macrophages were treated with CsA(500ng/ml) and recombinated CTLA4-Ig fussion protein at various concentration . Expression of cytokines such as TGF-01, VEGF, IL-8 was observed after 24h.Results1. Survival: the average survival of rejection group is 13. 17 days, which is no difference with survival of LacZ+ control group (12. 33 days).In comparison to it, survival of recipients in CSA-treaed group and CTLA4~Ig-transducted group are statistically prolonged (P<0.01)2. Expression of cytokines: IL-2 and IFN-y were highly expressed in rejection group as well as in LacZ group; expression of IL-10 in CTLA4-Ig-transducted group and CsA-treated group is higher than that of rejection and LacZ group. The cytokine level in serum of CsA treated group was in the middle between theose of rejection andCTLA4-Ig-transducted group.3. Infiltration of immune cells: infiltration of CD87 cell are marked in rejection group and LacZ group 3rd post operation, whereas CD8*T cell infiltrition in CTLA4-Ig-transducted group was less than that in CsA-treated group. Infiltration of macrophage was significantly faint in CTLA4-Ig-transducted group than in other groups.4. Cell apoptosis: the apoptotic index of rejection and LacZ group was significantly higher than those of CTLA4-Ig-transducted and CsA-treated group, whereas there was no difference between CTLA4-Ig-transducted and CsA-treated group. Apoptotic cells were able to be observed in any rejection grades, the mainly cell of apoptosis in every group were hepatocyte.A good positive correlation was shown between AI and acute rejection.5. Histological assessment: the severity of rejection reaction after day 3 was weaker in CTLA4~Ig-Transducted group and CsA-treated group than in rejection control and LacZ control group, on day 12 after transplantation, severity of rejection was lower in CTLA4-Ig-transducted group than in CsA-treated group.6. In vitro study: TGF-31 expressed by LPS-stimulated macrophages was decreased after CTLA4-Ig fussion protein treatment and was in the dose dependent manner. TGF-p 1 was increased af...
|
PDF Full Text Request