The Effect Of Bone Marrow Mesenchymal Stem Cells Derived From Recipients On Chronic Rejection Of Heterotopic Small Bowel Transplantation In Rat

PartⅠEstablishment of Chronic Rejection Model and Pathological Assessment of Small Bowel Transplantation in RatObjective:To establish the chronic rejection of rat small bowel transplantation model and to investigate the pathological changes after transplantation. Methods: Allogeneic heterotopic and orthotopic small bowel transplantation model were performed using microsurgery technology, and recipients received cyclosporine through subcutaneous injection at 0-14 days after surgery. Graft samples were harvested at POD90 and examineed by hematoxylin eosin staining and Verhoff's alcoholic hematoxylin ted. The function of transplanted gut was evaluated by ratio of Lactulose and mannitol and D-xylose absorption test. Results:(1)The recipients of heterotopic and orthotopic small bowel transplantation had a survival of more than 90 days. (2)The allografts of heterotopic small bowel transplantation showed histological features of chronic rejection at POD90. Histopathological analysis revealed distinctive abnormalities of allograft including changes of villous architecture, interstitial fibrosis, leukocyte infiltration, and intimal thickening. (3) The above histological changes were not observed in allografts of orthotopic small bowel transplantation. The allografts of orthotopic small bowel transplantation showed histological features of chronic rejection at about POD150, but lesions were mild compared with heterotopic grafts of POD90. (4)Most rat with orthotopic small bowel transplantation rat showed gradual weight loss after POD150, and it couldn't be reversed no matter cyclosporine was applied. When the weight loss more was more than 30%, the absorption and barrier function of orthopotic transplantative gut were decline compared with non-operative rats. Conclusion:(1)Chronic rejection occurred at POD90 in heterotopic small bowel transplantation model.(2)Histological features of chronic rejection in orthotopic small bowel transplantation model were detected after POD 150. (3)The surgical procedure of heterotopic small bowel transplantation is not complicated and surgical success rate is more than 85%.(4)Because typical histopathological features of chronic rejection exsits during the recipients survival time, the model of rat heterotopic small bowel transplantation is applied in the following research. According to pathological results, observative duration were confirm PODO-90 after transplant surgery.Part II The Research on Chronic Rejection of Heterotopic Small Bowel Transplantation in RatObjective:To extablish rat heterotopic small bowel transplantation model and to explore the mechanisms of chronic rejection. Methods:The recipients were divided into 3 groups including isogeneic control group, allogeneic control group and allogeneic transplantation with cyclosporine treatment group(CsA group). Graft samples were harvested at POD7, POD14, POD30, POD60 and POD90 and pathological examination was performed. The CD68 expression indicating infiltration of macrophages was determined by immunohistochemistry. In order to investigate the relationship of TGFβ1 and PDGF-C with chronic rejection, we used real-time quantitative RT-PCR and immunohistochemistry to detect the mRNA transcription and protein expression of TGFβ1 and PDGF-C. Results:(1)All of the recipients in the allogeneic control group died within 15 days after transplantation. (2)The leukocyte infiltration scores of CsA group were higher than those of isogeneic group from POD 14 to POD90. The mucous coat architecture scores and interstitial fibrosis scores of CsA group were higher than those of isogeneic group from POD30 to POD90. Intimal thickening scores of CsA group were significantly higher than those of isogeneic group from the beginning of POD60. (3)The expression of CD68 in CsA group continued to increase throughout the process of chronic rejection, and reached the highest level after POD60. (4)The mRNA and protein expression of TGFβ1 and PDGF-C in CsA group were significantly increased. The immunohistochemical results showed high expression of TGFβ1 in the interstitial and PDGF-C in the cytoplasm and cell membrane. Conclusion:(1)There was extensive infiltration of macrophages in the graft of CsA group at POD60, associated with high expression of TGFpl and PDGF-C. (2) The chronic rejection model of allogeneic heterotopic small bowel transplantation was successfully established at 60 days after surgery. The expression of CD68,TGFβ1 and PDGF-C were significantly increased in CsA group, which could be used to determine chronic rejection.PartⅢIsolation, Culturing and Identification of Rat MSCs from Bone Marrow in VitroObjective:To investigate the methods of the isolation, culture and identification of rat MSCs from bone marrow in vitro.Methods:One month-old male Lewis rats were sacrificed and tibias and femurs tissues were obtained. After the long bones were dissected, the marrows were flushed out with PBS buffer under aseptic condition and purified by percoll gradient centrifugation to get the mononuclear cells. After the mononuclear cells were cultured, the adherent cells were selected for serial subcultivation. The cells of the third generation in good growth condition was harvested and trypsinized to form cell suspension. The cells were counted and the growth curve was made. The cells of the third to five generation were harvested and the cell phenotypes were identified by flow cytometry. The cells of the third generation were induced to differentiate into osteoblasts. The induced cells were identified with alkaline phosphatase staining and alizarin red staining. The cells of the third generation were labeled by fluorescent dye of CFSE. Results:(1)The MSCs were spindle shaped, attached to the culture dish tightly after 72 hours' culture, and proliferated in the culture medium. After 21 days of primary cultivation, MSCs were nearly 100% confluent. (2)The 3th passage MSCs comprised a unique phenotypic population by flow cytometric analysis. The phenotypes were shown to be positive for CD29 and CD90 more than 96% and be negative for CD34 and CD45.(3)The MSCs formed aggregates or nodules and increased their expression of alkaline phosphatase, and calcium accumulation was evident after 3 weeks. Alkaline phosphatase staining and alizarin red staining confirmed the success of induction. (4)CFSE had high efficiency for labeling MSCs, the labeling index was over 100%.Conclusion:(1)MSCs are stem cells with strong proliferative activity and sustain self-renewal. They are pluripotent and could differentiate to a variety of cell types in some condition. (2)The methods of obtaining rat MSCs with density gradient centrifugation and adherence cultivation were convenient, and could provide immortalized rat MSCs lines for cellular transplant therapy. (3)CFSE staining is a feasible method for labeling MSCs with high efficiency. PartⅣStudy on the Effect of Bone Marrow Mesenchymal Stem Cells on Immunosuppression in VitroObjective:To explore the effect of mesenchymal stem cells derived from bone marrow using mixed lumphocyte culture on the immune response of splenic cells and the corresponding mechanisms.Methods:MSCs and mixed lymphocyte reaction cultures were set up. Splenic lymphocytes from F344 rat were used as reactive cells and labeled with CFSE. The study included 4 groups.Group 1 (the control group) included 5×105 reactive cells cultured. Group 2 were 5×105 reactive cells cultured with conA. Group 3 included 1×105 MSCs from Lewis rats and 5×105reactive cells. Group 4 included 1×105 MSCs from Lewis rats and 5×105 reactive cells cultured with conA as a stimulator. Cells in each group were cultured for 72 hours. The proliferation dynamics of CFSE labeled cells were detected by flow cytometry. In the same groups without CFSE labling, the CD4+ and CD8+ phenotypes of proliferating lymphocytes were determined with monoclone antibodies.Results:Compared with that in group 2, the proliferation of sub-peak in group 4 couldn't be detected. The positive expression of CD4+ and CD8+ were declined compared with that in group 1, but there was no difference in the ratio of CD4+ to CD8+. Conclusion:Bone marrow MSCs demonstrate significant immune regulatory effects on conA-stimulated T-lymphocyte culture. It might provide a remarkable immune suppression in organ transplantation to achieve better outcome in the future.Part V The Effect of Bone Marrow Mesenchymal Stem Cells Derived from Recipients on the Chronic Rejection in Rat Heterotopic Small Bowel TransplantationObjective:To observe the effects of MSCs derived from recipients'bone marrow on the chronic rejection of rat heterotopic small bowel transplantation. Methods:Sixteen Lewis rats bearing transplanted small bowel from F344 rats were randomly divided into 2 groups and all of them received cyclosporine A treatment. (1)Control group: 2mL 0.9%NaCl solution was injected through vena dorsalis penis at 24h after small bowel transplantation. (2)Treatment group:1×107MSCs in 2ml 0.9%NaCl solution was injected through vena dorsalis penis at 24h after small bowel transplantation. Graft samples were harvested on POD60 and examined by hematoxylin eosin staining and Verhoffs alcoholic hematoxylin ted. The graft infiltration of macrophages was determined by immunohistochemical staining. mRNA transcription and protein expression of TGFβ1 and PDGF-C were measured by real-time quantitative RT-PCR and immunohistochemistry, respectively. Two other small bowel transplantation models were established with cyclosporine A treatment. At 24h after transplantation, 1×107MSCs labeled with CFSE were injected through vena dorsalis penis, respectively. The two grafts and other self-organ were harvested on 7th day after transplantation to get frozen sections for observation of the fluorescence cells in grafts.Results:(1)Fluorescence cells concentrated in the interstitial of allografts. (2)The scores of mucous coat architecture, leukocyte infiltration, interstitial fibrosis and intimal thickening of treatment group were significantly less than those of control group at POD60.(3)The expression of CD68, TGFβ1 and PDGF-C in treatment group was significantly less than that in control group at POD60. Conclusion:Intravenous infusion with MSCs from recipients could inhibit the process of chronic rejection in rat heterotopic small bowel transplantation to some degree. The mechanisms involve the suppressive effect of MSCs on lymphocytic proliferation and on the expression of CD68,TGFβ1 and PDGF-C.