| Development of an animal model for tracheal xenotransplantation Objective To establish a simple guinea pig-to-rat tracheal xenotransplantation animal model, prepare for the advanced research on studying its immunological rejection characteristics and corresponding interventional approaches, and provide new insights into the clinical interventions to increase the tracheal procurement. Methods To establish a new short segment (5 rings) orthotopic tracheal xenotransplantation animal model by"adding-grafting technique", and observe the survival after operation, and the histopathological changes of the tracheal xenograph using optical microscope and electron microscope. The characteristics of immunological rejection were analyzed.Results 20 consecutive tracheal transplantation procedures had been performed by a single operator. The operation success rate was 100% (20/20). The duration of recipient procedure was (20.8±1.6) minutes. No complication observed. Post-transplantation survival durations ranged from shortest 8.0d to longest 12.0 d, with average (10±1.3) d. With the time increment, the tracheal lumen became narrowing down. Mucosal epithelium, submucosa, adventitia, and cartilage gradually showed various degrees of infiltration of lymphocytes, eosinophils, and macrophages, and cell apoptosis had been observed. Conclusions It is able to establish the short segment (5 tracheal rings) tracheal transplantation animal model by"adding-grafting technique". The histopathological changes post-transplantation agree with the characteristics of tracheal xenotransplantation immunological rejection. Therefore it is a simple, economic, robust, and repeatable animal model for transplantation research. PART 2Culture, Phenotype and Labeling of SD Rat's Mesenchymal Stem Cells in VitroObjective To investigate the methods of isolation,culture,phenotype and labeling of mesenchymal stem cells(MSCs) from SD rat in vitro, in order to prepare for the next study on the immunorejection of MSCs on tracheal xenotransplantation intervention. Methods MSCs were isolated and cultivated by adherent methods. The expression of cell surface antigen CD90 and CD45 was analyzed by flow cytometry in order to identify MSCs phenotype. The third generation of MSCs were labeled by DAPI. Results Primary cultured MSCs adhered to plastic surface within 48 hours and reached 90% confluence within 7-10 days. Flow cytometry showed that the positive rates of CD90 and CD45 MSCs at the third generation were 99.8% and 6.8%, suggesting that MSCs expressed CD90 but not CD45. Almost all of the MSCs after labeling by DAPI showed blue fluorescence under fluorescence microscope, suggesting DAPI labeling is sensitive and highly efficient for MSCs. Conclusions Adherent method is simple and easy to isolate and cultivate MSCs and it can become a routine protocol for culturing SD rat's MSCs. DAPI labeling can be used as an efficient method to label MSCs.PART 3 The Effect of Mesenchymal Stem Cells to Tracheal Xenotransplantation Immunological rejectionObjective Through the form of vein graft, injecting the mesenchymal stem cells(MSCs)into the recipient of guinea pig–to-rat tracheal xenotransplantation, further study the rejection reaction characteristics of tracheal xenotransplantation and MSCs's immune intervention function to it, aim to provide new ideas to solve the clinical problem of anti-rejection after tracheal xenotransplantation. Methods Experimental subjects were divided into 4 groups: A group (Autografting, n=17), B group (Xenotransplantation, n=17), C group(Xenotransplantation+ CsA, n=25), D group (Xenotransplantation+ MSCs, n=25).Recording receptor's survival time to observe the pathological changes in histology and degree of patency of the tracheal transplant, the expression situation of flow cytometry in peripheral blood CD4+,CD8+T lymphocyte, tracheal transplantation immunofluorescence examination of frozen specimens of IgG, IgM, C3 deposition, TUNEL detection of apoptosis in graft and calculate the apoptotic index (AI), peripheral blood biochemical analyzer creatinine (CREA) changes. Results (1) After xenotransplantation, the average survival time of the recipient in each group were (10.0±1.3) d, (17.1±2.7) d, (17.6±3.2) d in B, C, D group respectively. C , D group's survival time was significantly longer than B group (P <0.01). (2) Comparison of the pathological integral and degree of patency of the tracheal transplant in different group: compared to autotransplant group, xenotransplant groups showed significant increase in pathological points (P<0.01), and decrease in the degree of tracheal patency(P<0.01), these were due to acute rejection. The histopathological damages in groups C and D were less severe than group B, indicating the reduced immunological rejection. (3) Comparison of peripheral blood CD4 + ,CD8 + T lymphocyte's expression content: each group significantly increased than the autografting group after xenotransplantation, and kept in sustained increases over time, it illustrated that T cells were activated to involve in acute immunological rejection. B group showed the strongest increase, while C and D groups were less than it, indicating the suppression of T cell immune response.(4)Comparison of the immunofluorescence test results: no IgG, IgM, C3 deposition found in autografting group, different level of IgG, IgM, C3 deposition can be seen in each group after xenotransplantation, it illustrated that humoral immunity and complement system involved in acute rejection reaction; the complex deposition of IgG, IgM, C3 in B group was the most obvious, D group is lower than group B, if revealed that MSCs had a adjustment effcct to the humoral immune system and alexin system. (5)Comparison of the cells apoptosis in tracheal transplantation: AI from autografting group was significantly lower than the each group after xenotransplantation(P<0.01). B group had the lowest AI, and significantly differed from groups C and D (P<0.01). (6)Comparison of the peripheral blood creatinine (CREA): C group showed increase with time, and the remaining 3 groups were basically unchanged. Conclusions Both cellular and humoral immunity involved in the guinea pig-to-rat tracheal xenotransplantion acute rejection. Vein graft MSCs can significantly prolong receptor's survival time of the tracheal xenotransplantation, increase the patency of tracheal xenotransplantation, inhibit the apoptosis of cells, reduce the immune rejection. MSCs may interfere with immune rejection through the adjustment of immune cells and alexin system etc. factors. MSCs has a same anti-rejection effect with CsA, but it has not the side effects such as increase in infections and damage to renal function.
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