Research Of Inhibiting Cardiac Allograft Rejection In Mice By Interleukin-35 Modified Mesenchymal Stem Cells

Objective: To investigate the effect and anti-rejection mechanism of mesenchymal stem cells(MSCs)overexpressing IL-35 in mouse abdominal allogeneic heart transplantation,and to explore a novel effective approach to induce immune tolerance.Methods: 1.Adipose-derived mesenchymal stem cells were obtained using type I collagenase from mouse fat and identified by flow cytometry(FCM).2.IL-35 overexpression lentivirus vector(IL-35-LV)was made and infected to MSCs.Enzyme-linked immunosorbent assay(ELISA)was used to assay IL-35 expression in the cell supernatant.3.Mouse model of heterotopic abdominal heart transplantation was established by using Balb/c mice as the donor and C57BL/6 as the recipent.Mice were divided into IL-35-MSCs group,MSCs group,normal saline control group and syngeneic control group.Cardiac allograft survival time was observed,and cardiac samples were collected on day 5 after transplantation for pathology examination to grade the level of acute rejection.Western blot was used to detect the expression level of IL-17 in transplanted heart.IL-35 concentration in peripheral blood was measured by ELISA method at 5 days after transplantation.FCM was used to detect the changes of T cell subsets levels on day 5 after transplantation.Results: 1.Isolation of adipose-derived MSCs was successful.IL-35-LV infection to MSCs can secret IL-35,and the concentration of IL-35 in the supernatant of the cells was 265.8±16.650 pg/ml.2.Mouse model of heterotopic abdominal heart transplantation was successfully established.Cardiac allograft median survival time was significantly increased in MSCs group(10.67±1.211d)and IL-35-MSCs group(14.5±1.049d)when compare to the normal saline control group(6.17±0.753d)(P<0.01).Result of pathologic grading of acute rejection on day 5 after transplantation showed significant decrease in MSCs group and IL-35-MSCs group when compare to the normal saline control group (P<0.01).3.The result of ELISA demonstrated the concentration of IL-35 was 60.71±5.38 pg/ml in IL-35-MSCs group,and no IL-35 expression was observed in other groups.4.Compared with the normal saline control group,the Th1/Th2 and Th17 ratio of peripheral blood and spleen in MSCs group and IL-35-MSCs group was significantly reduced(P<0.01)and the percentage of CD4+ CD25+ T cells in the spleen was significantly higher(P<0.01).5.Western Blot showed the IL-17 expression in MSCs group and IL-35-MSCs group were significantly lower than that in the normal saline control group.Conclusion: 1.IL-35 gene modified MSCs can continuously and effectively express IL-35 in vivo and in vitro.2.Heterotopic abdominal heart transplantation in mice was a feasible model to study the rejection after tansplantation.IL-35-MSCs cell immune therapy resulted in the anti-rejection effect during heterotopic abdominal heart transplantation in mice and prolonged the survival time of transplanted heart.The possible anti-rejection mechanism is that IL-35 combined with MSCs can synergistically stimulate the generation of CD4+ CD25+ Treg,and induce balance offset of Th1/Th2 and decrease the proportion of Th17 cells to achieve extend survival time of the transplanted heart.Therefore,IL-35-MSCs cell immune therapy may be a novel effective approach to induce immune tolerance.