Effects And Mechanism Of Intrathymic Injection Of Donor BMSCs On Rat Groin Allograft Rejection

BackgroundVascularized composite allotransplantation(VCA) is currently one of the effective methods to cope with illnesses such as severe trauma, tumor excision and congenital malformation, which usually cause extensive tissue defects. However, all patients are generally required to take immunosuppressant for the whole lifetime after surgery and there is a quite high incidence of acute rejection episode which needs to be treated with pulse therapy of high-dose steroid and immunosuppressant. All these postoperative treatments can lead to serious side effects, thus greatly restricting the clinical application of VCA. As a consequence, to induce immune tolerance so as to reduce or even eliminate the usage of immunosuppressant becomes the most important thing for widely clinical application of VCA. Being the vital central immune organ of the body, thymus plays a crucial role in the induction and maintenance of both self-immune tolerance and transplantation tolerance and is the target spot of massive studies on transplantation immunity. There have been plenty of researches indicating that intrathymic injection of donor antigen can effectively induce recipient’s immune tolerance towards allotransplants. It has also been reported that allogenic bone marrow-derived mesenchymal stem cells (BMSCs) can site-specifically differentiate into thymic stroma cells in the thymic microenvironment of the recipient, which function critically in the positive selection and negative selection and contribute to the induction and maintenance of self tolerance. Therefore, intrathymic injection of donor-derived BMSCs into the thymus of recipient may help the BMSCs site-specifically differentiate into thymic stroma cells in the thymic microenvironment, thereby may be able to educate the host T cells and induce transplantation tolerance.ObjectiveBMSCs from donor BN rats were isolated, cultured and amplified in vitro and were intrathymic injected into the thymus of recipient Lewis rats. At the same time, the recipient rats were subjected to 4 Gy total-body irradiation with 60Coγ-rays. Influences of all these measures on the survival time of groin allograft were observed. Pathology examinations were also conducted on the allograft to compare the rejection between groups. The Treg(Regulatory T cells) ratio in recipients among groups were inspected via flow cytometry and reactivity of recipient splenic lymphocyte to donor antigen were compared by one-way MLR(mixed lymphocyte reaction) to preliminary study its mechanism on allograft rejection.MethodsBMSCs from BN rats were isolated by whole bone marrow adherent method and were purified and amplified with strict control of the time of culture medium change and tryptic digestion. The phenotypes of BMSCs in P3 generation were identified by flow cytometry and their ability to induce osteogenesis and adipogenesis were also identified. The recipient Lewis rats were randomly divided into four groups, with group A being the blank control group, group B the BMSCs group, group C the 4 Gy total-body irradiation group and group D 4 Gy total-body irradiation plus BMSCs group. All recipient rats accepted groin allotransplantation on day 0. PBS(Phosphate Buffered Saline) or donor BMSCs was intrathymic injected 14 days before allotransplantation respectively within group A and group B. The rats in group C were subjected to 4 Gy total-body irradiation 15 days before allotransplantation apart from intrathymic injection of PBS 14 days before allotransplantation. Rats in group D went through the identical irradiation with group C but were injected with BMSCs 14 days before allotransplantation. The mean survival time of groin allografts were observed and survival curves were plotted. Pathology examinations of allografts were carried out 7 days after allotransplantation. Change of Treg ratio in splenocytes and reactivity of recipient splenic lymphocyte to donor antigen were also detected by flow cytometry and one-way MLR at the end of rejection respectively.ResultsThe whole bone marrow adherent culture method can successfully purify and amplify substantial BMSCs and the P3 generation of BMSCs was positive for CD29、CD44、CD90, but negative for CD11b/c、CD34、CD45. When induced by adipogenic and osteogenic culture medium, BMSCs could differentiate respectively into adipocyte and osteocyte. There was no significant difference in the mean survival time of groin allografts between group B and group A(P>0.05), while the mean survival time of groin allografts in group D was significantly longer than that of group C(P<0.01), with a 3.4 days longer time. Compared with group C, The ratio of Treg in splenocytes enhanced significantly(P<0.01), and in MLR, the reactivity of spleen lymphocytes to donor antigen in group D were significantly lower(P<0.05). Besides, pathology examination demonstrated that groin allografts in group D showed less infiltration of inflammation cells with more integral skin structure and milder rejection in comparison to other groups.ConclusionThe whole bone marrow adherent culture method is a simple and easy way to isolate and culture BMSCs and can successfully amplify plenty of purified BMSCs. Intrathymic injection of donor BMSCs combined with 4 Gy total-body irradiation can significantly up-regulate Treg ratio in recipient splenocytes and reduce the reactivity of recipient lymphocytes to donor antigen, thus inhibiting rejection of allograft and significantly prolonging the mean survival time of groin allograft.