| Part one Isolation,proliferation,identification and function of lewis rat bone marrow mesenchymal stem cellsObjective:To isolate,proliferate,identify and research the function of lewis rat BMSC,Mixed lymphocyte culture and determination IL-2,IL-4,IL- 10 and INF-γ, provide experimental evidence for further use of BMSC-induced lower immune response of allogeneic liver transplantation.Methods:Lewis rat' BMSC were isolated and purified by the attachment culture method. The cell growth was detected and took a photo by an inverted microscope,and draw the growth curve.Identify the third generation of BMSC by CD34,CD44 immunohistochemistry staining.Analyse the phonotype of CD34,CD44,CD45,CD90 of BMSC by flow cytometer.Mixed lymphocyte culture,take a normal DA and Lewis rat spleen,purified T cells by nylon wool adsorption,as the stimulate cells and response cells,respectively.In response to cell culture plates by adding mitomycin C-treated 2.5×10~6 BMSC as the experimental group,the medium plate as a blank hole;the stimulate cells and response cells cocultured as the blank control;only add BMSC for the regulation of cell control.Calculate the rate of cell growth inhibition. In the primary culture of BMSC to 3d,5d,collect half of the culture medium,detect the level of IL-2,IL-4,IN-10 and IFN-γby ELISA method. Results:The cells showed active proliferative ability,single cell was narrow spindle-shaped. Cells arranged in spiral-shaped,mesh;double layer growth of the cells were multi-corner-type.The growth curve was similar to "S"-shaped.BMSC's expression of the CD34 was negative,while the relative specificity of BMSC marker CD44 expression was positive.Flow cytometry showed that the positive rates were 7.9%±1.3%,89.5%±4.5%,4.3%±0.5%,95%±3.8%of CD34,CD44,CD45,CD90, respectively.MLC show that in experimental group the absorbance A degree was 0.769±0.154,while in control group A degree 1.479±0.126,the inhibition rate was 48.05%,it showed that when BMSC and the Lewis rat T-cell ratio were 1:1,in the MLC system,T cell proliferation was obviously inhibited.In BMSC' culture medium, IL-2,IL-4,IL-10 and IFN-γwas not detected.Conclusion:We succeed in obtaining BMSC by the attachment culture method,identified it by morphology,immunohistochemistry,cell phenotype,functional activity ect,and confirmed that the isolateion and culture system is correct.In this study,it confirmed that BMSC has inhibited effect on T-cell,and in BMSC' culture medium,IL-2,IL-4, IL-10 and IFN-γwas not detected,which met the requirements for further experiments.Part Two Establishment of DA to Lewis orthotopic liver transplantation modelObjective:To establish a steady model of acute rejection model of orthotopic liver transplantation in inbred rats,so that to offer a technical platform for further experimental studies.Methods:Make the DA rat as donor and inbred Lewis rat as receptor,and adopt the improved Kamada "two cuff technique"to establish the model of orthotopic liver transplantation in the rats.Observe the situation after OLT;stat the survival time and the survival rate.Results:For the 20 rats with orthotopic liver transplantation,there are 17 rats were survival after OLT more than 48 hours.The operation time of donor and recipient were(30±8)minutes and(60±7) minutes respectively.The donor liver preparation was(15±2) minutes and the anhepatic phase was(21±3)minutes.The general successful rate was 85%.For 4 and 5 days after the operation,the rats showed jaundice,spirit of the poor,hard to open eyes,and all of them were dead in 13 days after the operation.Conclusion:According to the physiological characteristics,we had achieved some success by improving rat orthotopic liver transplantion technique.The acute rejection of DA to Lewis orthotopic liver transplantation model is stable and reliable.It can be effectively used to study allograft rejection and tolerance.Part Three Immune adjust effect on transplant rats with reinfused isogeneic bone marrow mesenchymal stem cellsObjective:To investigate the immune hyporesponsiveness and the character of immune adjustment by transplant rats with reinfused isogeneic bone marrow mesenchymal stem cells through portal vein,observe the protective effect on liver graft.Methods:Lewis rat' BMSC were isolated and purified by the attachment culture method. Take the third generateon BMSC to identification and phenotype analyse by flow cytometer(Part one).Make the DA rat as donor and inbred Lewis rat as receptor,and adopt the improved Kamada "two cuff techmque"to establish the model of orthotopic liver transplantation in the rats(Part two).Experimental classify for 4 group:group A is that Lewis rats received DA rat's liver without any treatment,which is the blank contrast group;group B is that the operation just like the group A,but with 10mg/kg·d of CsA treatment post surgery;group C is that Lewis rats received DA rat's liver and infusion of isogeneic BMSC in the amount of 2×10~6 cells during the operation,group D is that the operation just like the group C,but with 5mg/kg·d of CsA treatment post surgery.Observe the survival time and the situation after operation,all of groups were divided into four subgroups.Survival were observed of 6 rats in each group, recipients of a subgroup were sacrificed on day 4,7 or 10 respectively,serum levels of ALT,AST and TBIL were measured,the serum levels of IL-2,IL-4,IL-10 and IFN-γware tested by ELISA;apoptosis of hepatocyte and Th1 were monitored by FCM;the liver tissues were made into paraffin- embedded specimens to detect rat liver histological change and grade RAI(Banff scheme).Results:Compare with group A,the survival time of recipients in the group B,C,D was significantly prolong(P＜0.05);The level of ALT,AST and TBIL in group B,C,D gradually recoveried but group A was rapid advanced;The level of IL-2 and IFN-γin group A were higher than group B,C and D(P＜0.01);The level of IL-4 and IL-10 in group A were lower than group B,C and D at the same time(P＜0.01);Compared with group A,the apoptosis rate of hepatocyte decreased obviously after ROLT in group B,C and D(P＜0.05),at the same time the apoptosis rate of T-helper cells in liver in group B,C and D were lower than in group A.Group A's Banff score was significantly higher than group B,C and D(P＜0.05),and there was no significantly difference in the group B,C and D(P＞0.05).Conclusion:(1) BMSC improve rat liver graft function,reducee the pathology score of rejection,prolong the survival time.(2) BMSC can change the secretory volume of T-cell' cytokine,and inhibited the proliferate of lymphocyte.It also induce Th1 cell apoptosis in rat liver graft and reduce the apoptosis rate of heaptocytes and rejection after liver transplantation.(3) Subtherapeutic half dose of CsA act synergistically with BMSC to significantly inhibit acute rejection after liver transplantation.If we use BMSC, we can reduce the does of immuneosuppressant after liver transplantation,so that alleviate the side-effect of immuneosuppressant. |
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