Antirejection Effects On Liver Graft By Application Of Isogeneic Bone Marrow Mesenchymal Stem Cells Infusion Combined Donor's Spleen Tissue Transplantation In Transplant Rats

Part One Establishment of Acute Rejection Model of Orthotopic Liver Transplantation in RatsObjective:To establish a stable acute rejection model of orthotopic liver transplantation in rats,so that to offer a technical platform for further experimental studies.Materials and Methods:1 We underwent OLT skill training in closed population SD rats via two-cuff technique.2 Star the time of hepatectomy,liver pruning,anhepatic phase and the surgery in recipients;postoperative survival rate,survival time and causes of death were also observed.Results:1 The survival time of the rats were no more than 48 hours in skill training period,but the experiences of pre-surgery and post-surgery management were accumulated. 2 A successful rate of 85%was achieved in DA-Lewis Transplantation,3 cases were dead in 48 hours post-surgery,1 case died of hepatic inferior vena cava anastomotic bleeding,1 case died of air embolism and 1 case died of portal vein cuff fell off.3 17 cases got successful operation,the median survival time of 7 days was achieved.Conclutions:It has great difficulties in establishing a stable orthotopic liver transplantation model in rats,moreover,there were plenty of influencing factors.The surgery was success or not determined on masterly microsurgical technique,consummate pre- and post-surgery management and patience.Part Two 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,provide experimental evidence for further use of BMSC-induced lower immune response of allogeneic liver transplantation.Methods:1 Lewis rat BMSC extraction,cultivation and passage.BMC were harvested from 4 weeks aged lewis rat's femurs and tibias.After centrifugation,washing 3 times. Collected the precipitation cells and adjusted the cell concentration of 6×10⁵ cells/ml,placed it in 37℃,5%CO₂incubator.5d for the first medium change thereafter every 2-3d-for-1 medium change,then cells were seeded at 1.5×10⁴/cm² subculture,the culture conditions was the same as the original culture.Approach to the third generation of subculture in this way.2 Oberservation by microscope:Using the inverted microscope to photo and draw the growth curve.3 Identification and phenotype analyse:Identify the third generation of BMSC by CD34,CD44 immunohistochemistry staining.Analyse the phonotype of CD34, CD44,CD45,CD90 of BMSC by flow cytometer.4 Mixed lymphocyte culture.Take a normal DA and a 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 2.5×10⁵BMSC 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.5 Cytokines measurement of BMSC culture medium:In the primary culture of BMSC to 3d,5d,collect half of the culture medium,detect the level of IL-2,IL-4,IL-10 and IFN-γ,by ELISA method.Results:1 The third generation BMSC were single-layer shaped,closely attached with each other,arranged in a clear direction;single cells 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.2 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.It indicated that the isolated cells in line with the characteristics of BMSC.3 MLC showed that in experimental group the absorbance A degree was 0.769±0.154,while in control group A degree was 1.479±0.126,the inhibition rate was 48.05%,it indicated that when BMSC and the Lewis rat T-cell ratio were 1:1,in the MLC system,T cell proliferation was obviously inhibited. 4 In BMSC's culture medium,IL-2,IL-4,IL-10 and IFN-γwas not detected.Conclusion:1 It is Easy to harvest and isolate the BMSC by means of adherent screening2 In the experiments,detailed operation and sterile concept is the key to the success of cell culture3 In this study,morphology,immunohistochemistry,cell phenotype,functional activity, etc,confirmed that the isolation and culture system is correct which met the requirements for further experiments.Part ThreeThe Mechanism of Antirejection Effects on Liver Graft by Application of Isogeneic Bone Marrow Mesenchymal Stem Cells Infusion Combined Donor's Spleen Tissue Transplantation in Transplant RatsObjective:To study the mechanism of immune hyporesponsiveness and antirejection effects of allograft rejection induced by isogeneic bone marrow mesenchymal stem cells infusion combined donor's spleen tissue transplantation, observe the protective effects on liver graft.Methods:1 Establish the DA-Lewis liver transplantation model(part one),isolation,proliferation, identification of BMSC from Lewis rats(part two)2 Experimental classification 96 experimental rats were randomly divided into 4 group,24 in each group(1) Control group(A group):Lewis rats received DA rats' livers without any treatment.(2) CsA group(B group):Lewis rats received liver transplantation with 10mg/kg of CsA treatment post surgery.(3) BMSC group(C group) Lewis rats receive liver transplantation and infusion of isogeneic BMSC in the amount of 2×10⁶ cells during the operation.(4) BMSC combined with spleen tissue transplant group(D group):Lewis rats receive liver transplantation and infusion of isogeneic BMSC in the amount of 2×10⁶ cells and transplantation of the DA rats' spleen tissue(25%of the whole spleen)in greater omentum.3 Survival were observed of 6 rats in each group,blood levels of ALT,AST,TBIL,IL-2,IL-4,IL-10and IFN-γwere determined and liver pathological changes were observed on day 3,7and 10 post-operation,the apoptosis rate of T-helperl cells and hepatocytes were also been dectected by flow cytometry,the level of chimerism in lewis rats was detected at day7.Also,the transplant spleen tissue pathological changes were observed.Results:Compared with group A,survival time of the recipients in group B,C was significantly prolonged(P＜0.05),D group had the longest survival time;The level of ALT,AST and TBIL in group A were rapid advanced but gradually recoveried in other groups.The level of IL-2 and IFN-γin group A were higher than other groups(P＜0.01),and group D vs group C,D(P＜0.05).The level of IL-4 and IL-10 in group A were lower than other groups,group D achieved the highest leverl(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 C and D were lower than that in group A and B.The chimerism level in group D was the highest among the four,group A's Banff score was significantly higher than groupB,C and D(P＜0.05),among which there was no significantly difference(P＞0.05).Conclusion:1 BMSC improve rat liver graft function,reduce the pathology score of rejection, prolong the survival time.2 BMSC induce Th1 cell apoptosis in rat liver graft and reduce the apoptosis rate of hepatocytes and rejection after liver transplantation.3 Spleen tissue transplantation increase the level of chimerism in recipients,prolong the survival time of liver graft and play a synergistic role in anti-rejection caused by BMSC in rat liver transplantation.4 Allogeneic spleen tissue had regeneration in recipients'.