Immunomodulatory And Liver Regeneration Effects Of Adipose-derived Mesenchymal Stem Cells From Donor In A Rat Orthotopic Small-for-size Liver Transplantation Model

Introduction:At present, allogeneic orthotopic liver transplantation was the only effective means to cure the end-stage liver disease, but its development was limited by the shortage of organs。In order to expand the supply of organs and partially overcome the grafts shortage, split liver transplantation and living-related liver transplantation have played a leading role。But the safety matching of donor and receptor was still the toughest problem on the current。For instance, the liver was supposed to cut as small as possible in order to keep the safety and the less complication incidence of donor in living-related liver transplantation. However, the benefits of them were limited in adult recipients when the volume of the grafts was small. The small-for-size grafts were hard to tolerant the damage of ischemia reperfusion and the immunologic rejection simultaneously. Once the hyperplasia of the hepatic cells couldn't stand against the damage given by the factors mentioned above, the Small-for-size grafts would be lost。This was the antinomy which the clinician at work on liver transplantation had to face。To find out the method which not only could make small-for-size grafts regenerate quickly, but also could lower immune rejection or lead to immunological tolerance was the problem waiting to solve in living donor liver transplantation at present。The research about liver regeneration mainly concentrates in the factors of regeneration. But the half-life of outside source liver regeneration factors was very short and the factors needed to be input again and again in order to have its possibility effect. However, various hepatic growth factors have not been commercialized production. The decrease of hepatic cells was absolute in small-for-size liver transplantation. The hope that liver cell could regenerate rapidly and effectively relying on the various hepatic growth factors was extremely difficult.Part ⅠLiver transplantation in rats using30%small-for-size grafts and the surgical techniques improvementBackground:The techniques of partial liver transplantation, using a living donor graft, expanded the supply of organs and partially overcame the grafts shortage. But these benefits were limited in adult recipients when the volume of the grafts was small. To establish a model of small-for-size liver transplantation, using a simple and effective way, was the basis for the study mentioned above.Objective:To explore a simple and effective way of establishing a30%small-for-size liver transplantation in rats.Methods:280Spraque-Dawley rats were selected as the donors and recipients to establish30%Small-for-size orthotropic live transplantation using two-cuff techniques. Animals were divided into two groups depending on the techniques modified or not.60pairs of rats were divided into before modification group, using the way according to reference10to15and the median lobe of the liver as graft. These60pairs of rats were divided into two groups again depending on hepatectomy in vivo or in vitro. Group Ⅰ, performed hepatectomy in vivo before liver irrigation; group Ⅱ, performed hepatectomy in vitro after liver irrigation.80pairs of rats transplanted using the way of improvement by us were divided into modification group (group Ⅲ), in which hepatectomy was performed in suit after liver irrigation, the median and right lobes of liver were used as graft, body weights of donor were100-120g less than those of recipients, two-cuff technique and bile duct stent techniques were improved. Time of operation, survival and technical complications were compared among these groups.Results:The time (min) of hepatectomy and graft harvest were significantly shorter in group Ⅲ than those in group Ⅱ and Ⅰ (9.0±0.7vs.15.0±1.5vs.15.6±1.4, P=0.005;56.6±3.4vs.73.6±2.3vs.74.6±3.0, P=0.002). The cold ischemia time in group II was significantly longer than those in group Ⅰ and Ⅲ (81.0±2.2vs.62.6±3.1vs.59.8±3.2, P=0.001). The time of anhepatic and recipient operation were not significantly difference among them. The incidence of bleeding, bile leakage IVC stricture, graft less perfusion and gas embolism were significantly less in modification group than those in before modification group. The rats in modification group had a higher transplanting successful rate (92.5%), more7-d and14-d survivors post operation (62.5%&50.0%) and longer median survival time(14d) than those in before modification group(P<0.05).Conclusion:The way of modification by us was a more effective and simple for establishing a30%small-for-size liver transplantation in rats with higher transplanting successful rate and survival rate but fewer complications after operation. Part IIIsolation, proliferation, Identification and differentiation into hepatocyte-like cells of rat adipos-derived mesenchymal stem cellsObjective:To explore the rat AMSCs acquisition method in vitro,biological characteristics, the capacity of differentiation into hepatocyte-like cells in vitro.Methods:We fetch inguinal fat of male SD rats to primary culture, observe cell growth and cell morphology, depict cell growth curve, take the fifth generation cell and flow cytometry identified this cells and determine cell purity. Preparation of conditioned induced medium in vitro, use the fifth generation cell to prepared cell climbing film, add induced medium culture cell, after12,24d immunohistochemistry detected cell Alb and CK18.Results:①The cell covered the bottom about10days after primary culture, the cell were fibroblast-like or vortex-like, with the more passage times, the cell morphology more single, cell growth curve like "S". The third to ninth generation cells had the strongest proliferation.②By flow cytometry to detect cell surface markers, CD44expression rate about98.5%, CD90expression rate about92.6%, CD49d expression rate about90.5%, CD29expression rate about73.1%, CD45expression rate about0.3%, CD34expression rate about0.6%. Through the combination between them surface markers confirm this cell is AMSCs, and has high purity.③Cell climbing film began morphological changes from long fusiform into edge shape or oval through5days induce. Some cell appeared dual-core, this is unique to liver cells12days after induced. Alb and CK18expression positive on12,24days.Conclusion:①AMSCs can be got by a special cultured method from the adipose tissue, and cultured in vitro can be passaged for many times, a lot of amplification, while maintaining its biological characteristics. They had a very strong proliferation, and the third to ninth generations had strongest proliferation.②Confirm these cell is AMSCs by flow cytometry detected a variety of cell surface antigens and the combination of many different surface antigens, and these cell has high purity.③MSCs can differentiate into hepatocyte-like cells in a suitable culture medium, and express the liver cell antigen; it has the part function of liver cells. Part IIIMechanism of immune hyporesponsiveness and liver regeneration induced by the Adipose-derived Mesenchymal Stem Cells from donor in a rat30%Small-for-Size Liver Transplantation modelObjective:To observe the effect that AMSCs induced immune response in rat model of30%small-for-size liver transplantation, while promoting transplant liver regeneration, and Preliminary investigate the mechanisms. Provide some basis for the further extensive and in-depth study, and the application of clinical.Methods:We transplanted the AMSCs of the male Lewis rats, obtained as the method in part one, into the rats underwent the30%small-for-size liver transplantation as the method in part two. The rats were divided into group A (comparison group), group B (FK506group), group C (AMSCs single input group) and group D (AMSCs preoperative and postoperative multiple input group). The survival and the rejection in different groups were observed. The serum level of liver enzymes, IL-2, INF-γ, IL-4, IL-10and TGF-β1was examined by ELISA. The variation of PCNA,Ki-67,TGF-,β1, NF-κB,ICAM-1,IDO and apoptosis in the grafts was detected by immunochemical methods. The Y chromosome (SRY) in the grafts of group C and D was detected by hybridization in situ. The one-way mixed lymphocyte reaction (MLR), recombined IL-2reverse assay of one-way MLR and adoptive transfer assay were performed in group D. Results:①The median survival time of rats was8±0.55d in group A,14±1.1d in group B,10±1.1d in group C and21±2.2d in group D respectively. The survival time of rats in group D was longer than that in group A to C significantly.②The serum level of liver enzymes increased after the operation in all groups significantly. It rose continuously in group A to C and decreased in group D as time goes by. The serum level of liver enzymes was significantly lower than that in group A to C on the7th day after operation.③The serum level of the cytokines increased after operation in all groups significantly. The level of IL-2and INF-γ raised continuously and rapidly in group A and C as time goes by. But it rose slowly in group B and D. Furthermore, it decreased in group D on the7th day after the operation. The level of IL-4and IL-10changed oppositely in all groups against that of IL-2and INF-γ. The level of TGF-β1increased after operation in all groups significantly. It rose continuously in group A and C as time goes by. It was tend to rise slowly in group B on the5th day, but rose quickly again on the7th day after the operation. In group D it was decreased gradually as time goes by. Its level was lower in group D than it in group A to C on the7th day after the operation.④The immunochemical examination of grafts:The IDO conveyed in the cytoplasm and its level was not changed significantly in all groups as time goes by after the operation. The ICAM-1conveyed in the membrane and the TGF-β1in the cytoplasm. Their level raised continuously in group A to C as time goes by. They decreased gradually on the5th day after the operation and were lower on the7th day in group D than theirs in group A to C. PCNA and Ki-67conveyed in the nucleus. Their level decreased rapidly after the operation in group A to C as time goes by. Otherwise, their convey always maintained a high level after the operation. The number of apoptotic bodies in the grafts in group D was less than that in group A to C.⑤The double positive cells of the SRY hybridization in situ(brown nuclei) and Alb/Ck18immunohistochemistry (purple/violet blue cytoplasm) would be observed in the grafts in group C and D on the5th and7th day after the operation. They increased gradually in the number in group D as time goes by and decreased quickly in group C. The number of the cells in group D was higher than that in group C significantly on the5th and7th day after the operation.⑥The score of Banff was grade0to1in all groups on the1st day after the operation. It was grade2in the group A and C, grade1in the group B and grade0to1in group D on the5th day after the operation. It was grade3in the group A to C but still grade0to1in group D on the7th day after the operation.⑦The SI%of one-way MLR answered to donor strain(Lewis) and unrelated strain(SD) was32.7±3.7%and39.7±12.3%respectively. There was not significantly difference between them. The SI could be reversed by IL-2similarly between them. In vivo adoptive transfer assay was not observed in group D.Conclusion:The way of AMSCs infusion repeatedly on the Pre and post liver transplantation using small-for-size graft in rats could inhibit the acute rejection while promote the graft regeneration through a variety of mechanisms. It would have the liver function recovered faster and prolong the survival time of the rats. The AMSCs infused could colonized in the graft and differentiated to the hepatocyte-like cells but would not induce the immune tolerance.