| Part 1:Establishment of the models of orthotopic liver transp lantation and acute rejection of allograft in ratsObjectives:To establish a stable model of orthotopic liver transplantation and acute rejection of allograft in rats.Methods:20 cases of Sprague-Dawley (SD) rat to SD rat isograft OLT and 20 cases of DA rat to Lewis rat allograft OLT were done. The operation was made following the modified Kamada's two-cuff technique, after fully exposing the first portal, perfuse through the portal vein (PV) without turning, we use the double traction to install PV and inferior vena cava (IVC) cuff. 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:donor harvest time (42.2±4.5) min, recipient operation time (45.5±3.5) min, anhepatic phrase (15.0±2.1) min,24h survival rate 90.0%, 1w survival rate 85.0%. In isograft group,1 cases died during the operation,1 cases died of complication during the observation,and the survival time (55.75±3.20) d; on POD7, liver function and graft pathology were normal, RAI score (1.75±0.70). In allograft group,3 cases died during the operation,1 cases died of complication during observation,and the survival time (10.25±2.57) d; on POD7, serum levels of ALT, AST and TBIL significantly increased, and graft pathology show severe rejection, RAI score (7.85±0.72).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.Part 2:Isolation and culture of donor bone marrow mesenchymal stem cellsObjective:To isolate and culture rat bone marrow mesenchymal stem cells in vitro.Methods:We culture the BMSCs by density gradient centrifugation and adherent, then use inverted microscope for cell morphology, flow cytometry to test cell phenotype, and induce BMSCs differentiate into bone, fat and cartilage cells in vitro. Results:Density gradient centrifugation and adherent can effectively isolate BMSCs. all cells were fibroblast-like under microscope; flow cytometry show:CD44 and CD29 positive, CD34 and CD44 negative. It indicated that the cultured cells were neither hematopoietic stem cells nor fibroblasts, and these cells had multiple differentiate potential.Conclusions:(1) Density gradient centrifugation and adherent methods can effectively isolate mesenchymal stem cells from rat bone marrow;(2) We confirmed the characteristics of mesenchymal stem cells by morphology, immunophenotype and differentiation potential.Part 3:Studis of Mesenchymal stem cells inhibiting immunological reaction in vitroObjective:To explore the roles and mechanisms of MSCs in T lymphocyte immune response of allogeneic mixed lymphocyte reaction in vitro.Methods:Establish the mixed lymphocytes reaction system, the reaction volume is 200?L. The stimulator is DA rat spleen T cell, the reaction cell is Lewis spleen T cell, and the cell number ration is 1:1(1 ×105/50ul). There were 4 groups, group A:control group; group B:add 1×103/1ul DA rat MSCs in MLR system; Group C:add 1×104/10ul DA rat MSCs in MLR system; Group D:add 1×105/100ul DA rat MSCs in MLR system. After 5 days'culture, added 3H-TdR, then measured CPM value by liquid scintillation and measured IL-2, IL-4, IL-10 and IFN-?levels in supernant by ELISA.Results:MSCs can inhibit T lymphocyte proliferation in the MLC system, and showed dose-dependent; levels of IL-2 and IFN-y were the highest in the control group, they decreased with the number of MSCs increase (P<0.05), the differences were significant; levels of IL-4 and IL-10 were the lowest in the control group, they increased with the number of MSCs increase (P<0.05), the differences were significant.Conclusions:(1) MSCs can decrease IL-2 and IFN-?secretion by Th1 cells, increase IL-4 and IL-10 secretion by Th2 cells, it was associated with the number of MSCs;(2) MSCs can inhibit the T lymphocytes proliferation in MLC, which was probable associated with regulating Th1/Th2 reaction balance. Part 4:Impacts of MSCs in rat acute rejection after liver transplantationObjective:To explore the roles and mechanisms of MSCs in rat acute rejection after liver transplantation.Methods:The transplant models were made following the modified Kamada's two-cuff technique with DA rats as donors and Lewis rats as recipient. There were 6 groups, group A:sham operation group, n=6; group B:control group, n=12; group C, Rapamycin group, with Rapamycin (2mg/kg/d) taken by oral for 14 days from POD1, n=12; group D, MSCs group, with MSCs injection through portal vein, n=12; group E, MSCs+Rapa group, with MSCs injection through portal vein and Rapamycin (2mg/kg/d) taken by oral for 14 days from POD1, n=12. Groups B, C, D and E divided into two subgroups (n=6), one subgroup were used for observing survival time, and the other subgroup were used for experiments. We tested Serum levels of ALT, AST, TBIL; pathological examination by HE staining; serum levels of IL-2, IL-4, IL-10 and IFN-y by ELISA; spleen CD4+CD25+Foxp3+Treg cell ratio by flow cytometry.Results:Compare with group D, survival time reduce in group B and C(P<0.05), but it prolonged in group E(P<0.05); on POD7, serum levels of ALT, AST and TBIL significantly increased in group B and C(P <0.05), and they significantly decreased in group E (P<0.05); RAI significantly increased in group B and C (P<0.05), and it significantly decreased in group E (P<0.05); compare to group B and C, serum levels of IL-2 and IFN-y decreased in group D and E (P<0.05), and it significantly decreased in group E (P<0.05). serum levels of IL-10 significantly increased in group E (P<0.05), and serum levels of IL-4 had no significant difference; the ratios of CD4+CD25+Foxp3+Treg in spleen cells, it significantly increased in group C, D and E (P<0.05), compare to group C and D, it significantly increased in group E(P< 0.05)Conclusions:(1) MSCs can improve liver function, reduce rejection, prolong survival time;(2) MSCs can change T cell cytokines secretion and ratios of CD4+ CD25+Foxp3+Treg, reducing the rejection;(3) MSCs and Rapamucin had synergistic effect probably on inhibiting the immune rejection. |
PDF Full Text Request