| Part1:Establishment of the models of rat orthotopic liver transplantation and acute rejection of allograft in inbred ratsObjectives:To establish a stable model of orthotopic liver transplantation and acute rejection of allograft in rats.Methods:100cases of rat OLT were performed by modified Kamada’s two-cuff technique in SD rats, followed by20cases of SD to SD OLT and20cases of DA to Lewis OLT in rat. After the operations, we exam the liver function and survival time, the grades of acute rejection in allografts were judged according to Banff scheme.Results:Of the last40cases, the duration of anhepatic time was16.76±2.86min, the successful rates of operation and1w survive rates were90.25%and80.00%, respectively.In isograft group,the survival time (41.27±6.25) d; on POD7, liver function and graft pathology were normal, RAI score (1.76±0.75). In allograft group, the survival time (9.66±1.38) d; on POD7, serum levels of ALT, AST and TBIL significantly increased, and graft pathology show severe rejection, RAI score (7.70±0.81).Conclusions:(1)The modified Kamada’s two-cuff technique can be used to made a stable model of the rat orthotopic liver transplantation for the advanced research in the future;(2)DA'Lewis allograft liver transplantation in rat is a stable model for acute rejection after liver transplantation. Part2:The construction of recombinant human Foxp3gene lentiviral vector and its expression in MSCsObjective:Preparation of lentiviral vector carrying human Foxp3gene expression in MSCs.Methods:The Foxp3gene was acquired by PCR from hela cells with a appropriate primer,the product of PCR were combined to produce sticky ends with FUGW carrier by standard procedure. The Foxp3gene andFUGW carrier were co-transformated into the Stb13cells to produce recombinant lentiviral granule(Foxp3-FUGW). Then, the recombinant lentiviral granule were transfected in293FT cells to product packaging recombinant lentiviral granule. After identified, the desired recombinant Lentivirus were purified by density gradient ultracentrifuge and titrated.Finally,the lentiviral granule was transfected into MSCs(stem from DA rats).Results:The hela cells were identified correct by sequencing and the recombinant lentiviral plasmid(FUGW) containing target gene were selected for further study. Packaging recombinant lentiviral granule with high pure and titer(2×1010IU/ml) were generated after density gradient ultracentrifuge.Conclusion:Success in construction of recombinant lentiviral vectors and obtaintion packaging recombinant lentiviral granule with high pure and titer(2×1010IU/ml).The Foxp3-FUGW was able to infect and express in the MSCs. Part3:The impact of MSCs stably expressed Foxp3on immunomodulatory effectsObjective:To explore the roles and mechanisms of MSC-Foxp3in CD4+T cells immune response of allogeneic mixed lymphocyte reaction in vitro.Methods:Established the mixed lymphocytes reaction system, the reaction system volume was200μL. The stimulator was spleen T cells from SD rat, the reaction cell was spleen T cell from Lewis rat, and the cell number ration was1:1(1×105/ul).There were4groups, group A:add100ul PBS in MLR system; group B:add100ul MSCs (1×105/ul) in MLR system; Group C:add100ul MSC-Foxp3(1×105/ul) in MLR system; Group D:add100ul MSC-Foxp3(1×105/ul)+Transwell in MLR system.All the MSCs stem from DA rat. After5days’culture, added3H-TdR, then measured CPM value by liquid scintillation and measuredTNF- α、INF-γ、TGF-β and IL-10levels in supernant by ELISA.Results:Compared with MSCs, the MSC-Foxp3could more inhibited proliferation of the CD4+T cells(P<0.05), reduce the content of TNF-α、IFN-γ and increase the content of TGF-β、 IL-10(P<0.05).Blocked the direct contact between MSC-Foxp3and T cells by the Transwell experiment,MSC-Foxp3+Transwell could recovery the proliferation of the CD4+T cells significantly(P<0.05), increase the content of TNF-α、IFN-y and reduce the content of TGF-β、 IL-10(P<0.05).Conclusion:(1)Compared with unmodified MSCs,MSCs over-expression of Foxp3had more powerful immunomodulatory capacity;(2)Immunosuppressive mechanisms of MSC-Foxp3might be similar to that of MSCs,including cell-cell contact and soluble factors possibly. Part4:The impact of MSCs-Foxp3on preventing allograft in acute rejection following rat orthotopic liver transplantationObjective:To explore the roles and mechanisms of MSC-Foxp3in acute rejection following rat orthotopic liver transplantation. Methods:Orthotopic liver transplantation was performed following the modified Kamada’s two-cuff technique with DA rats as donors and Lewis rats as recipient. The experimental animals were divided into4groups randomly, group A:sham operation group, n=8;Group B:received unmodified grafts, intravennous injectionPBS buffer after transplantation at the time of24,as control group, n=16; Group C:intravennous injection MSCs(stem from DA rat) after transplantation at the time of24h, n=16; Group D:intravennous injection MSC-Foxp3(stem from DA rat) after transplantation at the time of24h, n=16.Groups B, C and D were divide into two subgroups, one subgroup were used for observing survival time, and the other subgroup were sacrificed on day7after transplantation used for experiments, serum levels of ALT, AST and TBIL were measured; frozen sections made of part of the liver tissues were observed GFP expression by fluorescent fiberopic, the rest of liver tissues made into paraffin-embedded specimens to detect rat liver histological change and grade RAI(Banff scheme);serum levels of IL-2,IFN-y,IL-10and TGF-β was tested by ELISA; spleen CD4+,CD8+,CD4+CD25+Treg ratio was tested by flow cytometry.Results:MSC cells that expressed Foxp3after viral infection, which scattered in the liver,were observed through fluorescent fiberopic; Compare with group C, survival time reduced in group B (P<0.05), and it prolonged in group D (P<0.05); on POD7,serum levels of ALT, AST and TBIL significantly improved in group B (P<0.05), and they significantly decreased in group D(P<0.05); RAI significantly increased in group B (P<0.05), and it significantly decreased in group D (P<0.05);compare with group C, serum levels of IL-2and IFN-y significantly increased in group B (P<0.05),and they significantly decreased in group D(P<0.05),serum levels of IL-10and TGF-β had the opposite result, the differences were significant; as to the ratios of CD4+in spleen cells, it significantly decreased in group B, and they significantly increased in group D (P<0.05),CD8+also had the opposite result, the differences were significant; the ratios of CD4+CD25+Treg in spleen cells, it significantly increased in group D<0.05),and they significantly decreased in group B (P<0.05)Conclusion:(1)The MSC-Foxp3could prevent allografts from acute rejection in rat OLT, and the mechanism might be associated with increased proportion of Treg cells and cytokine secretion;(2)The MSC-Foxp3could enlarge the immunomodulatory function of MSCs and possess more poweful immunosuppressive function than them. That might be partly a result of Foxp3transcription, gene. |
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