| Part One Establishment of Orthotopic Liver Transplantation Model in RatsObjective:To investigate the establishing methods of orthotopic liver transplantation model in rats and provide a stable basic model for liver tansplantation research. And then we deal orthotopic liver transplantation model in rats with DA-Lewis and SD-SD, to take knowledge of the high lever of immunity rejection in DA-Lewis in survival time and pathology.Methods:Using Kamda two-cuff-technique to establish rat orthotopic liver transplantation model. During pre-experiment, SD rats were exceuted with orthotopic liver transplantation, while in formal orthotopic liver transplantation experiment, DA and Lewis rats were used as donors and recipients respectively. After the operation, recipient survival rate and liver function and pathology were observed.Results:Orthotopic liver transplantation models were successfully established in248rats. The main problems in the training phase were to overcome the problem of anesthesia, vascular anastomosis, to shorten the time of the anhepatic phase, to improve the management after the anhepatic phase. Analyzing50model standard operations, donor operation time was35.78±5.78min,warm ischemia time was0.5±0.22min, donor truing time was15.13±3.45min, recipient operation time was40±10.5min, the time before anhepatic was37±7.11min, anhepatic time was21.33±3.65min, the time after anhepatic is13.98±3.83min,operation success rate was97.3%. At the formal examination, the model from DA to Lewis showed high lever of immunity rejection by survival rate shortened and liver function increased rapidly and severe pathology.Conclusion:To establish methods of orthotopic liver transplantation model in rats successfully, operators must have the process of orthotopic liver transplantation the techniques extent microsurgery,the knowledge of rat abdomen anatomy and the manipulate of aseptic surgery, and lastly hard training for half of a year was inevitable. Key factors for successful operation include right liver infusion methods, rigorous peritoneal hemostasis,skilled microvessel suture technique, shortened anhepatic time, improved fluid therapy, corrected acid and alkali balance during operation and rewarming after operation. The model of liver transplantation from DA to Lewis can successfully replicate the high rejection in rat liver transplantation model. Part Two the research of the immunologic characteristics of the hepatic-like cells differentiationed from adipose tissue-derived stem cell in vitroObjective:To investigate the effect of the improved method of Monoclonal screening which was used to purification adipose tissue-derived stem cells(ADSCs) in the original generation culture.To differentiated the rat ADSCs into the hepatic-like cell (iPS) in vitro by procedure induction. The rat adipose tissue-derived stem cells were differentiated into the hepatic-like cells in vitro and the immunologic characteristics of iPS were investigated.Methods.After isolated the Lewis rat ADSCs with the digestion centrifuge process, The ADSCs was taken with the limited density dilution method cloning and condition-culture medium screening in the original generation culture.The group with clone screening ADSCs was taken as the experimental group, without clone screening as the control group, comparied the cellular appearance, growth curve and the cell doubling generation time; the surface marks CD49d, CD34, CD44of ADSCs were determined by flow cytometry.The rat ADSCs was differentiationed into iPS in the procedure culture system by tris-step including FGF-4,FGF-α,HGF,dexamethasone and OSM. The ALB,AFP, CK18mRNA were determined by RT-PCR and Urine and transferring Ammonia were monitored during inducing differentiation periods. The ALB was determined by fluorescence immunohistochemisty at last. To detect the Immunoregulation function of rat ADSCs and the iPS differentiationed from ADSCs by mixed lymphocyte culture of the system of the DA/Lewis spleen cell, and the effect of immuno-regulation on IL-2and IL-10of spleen cell were determined by ELISA analyze. Result:The appearance of ADSCs which undergone clone screening has been homogeneous. The appearance were the doubling time was shorten, the change of growth curves was not obviously after many generation, growing-power after long-term culture was vigorous and stable, and the surface marks CD49d, CD44were high expressed while the surface marks of CD34was low in both the control group and the the experimental group examined by FCM.Rat ADSCs was successfully differentiationed into iPS by the21d culture system in vitro. The expression of ALB, AFP and CK18mRNA was determined fortified by degrees at7thd,14thd and21thd. The protein ALB was determined by immunohistochemisty. It was occurred and persistenced of Metabolism of ammonia and urea synthesis after9th-12th d. It was shown that both Lewis rat ADSCs and iPS can down-regulate the immunologic reaction of the co-culture of DA and Lewis rat spleen cell indifferently, and the immuno suppression effect was enhanced by concentration of Lewis rat ADSCs and iPS.Conclusion:The clone screening purification method is effective,economical purification method of ADSCs. Rat ADSCs was successfully differentiationed into iPS in vitro after procedure-induction. The method of induction can provide a valid and reliable source of seed cells for hepatocytes transplantation, the clinical treatment of liver tissue engineering. Rat ADSCs was successfully differentiationed into iPS in vitro, the iPS have the characteristics both function of Liver cell regeneration and immunosuppression effect. Part Three the influence to the immunity rejection of the orthotopic liver transplantation model in rats induced by the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells.Objectives:After injected the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells by portal vein in the process of the model of the orthotopic liver transplantation in rats of the model with high-lever rejection (DA-Lewis). To investigate the homing position in the rat liver, and the influence on the liver function the degree of immunity rejection survival time. The safety, efficiency and mechanism of the hepatic-like cells differentiation from adipose-derived mesenchymal stem cells in the orthotopic liver transplantation model in rats were discussed.Methods:1. The model of the orthotopic liver transplantation in rats with high-lever rejection (DA-Lewis) was established with Kamada two-cufft technique, divided the DA and Lewis rat into4group(31recipient/group) randomizing,1) The group A (reject group):operation without any other management2) The group B (the immunosuppressant group) operation and take FK506capsule orally (0.15mg/kg/d by Gastric lavage qdX14d begin after the second day of operation)3) The group C:operation and injected the adipose-derived mesenchymal stem cells by portal vein in the process of operation.4) The group D:operation and injected the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells by portal vein in the process of operation. 2. The general condition of the luster of eyes,rat hair,activity and mental state of each experiment rat were observed and recorded;6rat were left to observe the survival time each group;3.6rats were killed at the4th,7th,10th,14th day after operation each group. The liver function and cytokine (IL-2, INF-y and IL-10and IL-4) were detected. Cytokine were detected by the ELIS A method.4. Brdu was used to mark the adipose-derived mesenchymal stem cell and hepatic-like cells differentiationed from adipose-derived mesenchymal stem cell in vitro. Identification of the mark of Brdu by fluorescence immunohistochemical, and detected the homing position of the cell in vivo marked by Brdu with fluorescence immunohistochemical methods.5. The hematoxylin-eosin staining of rat liver was taken to observe the rejection activity index.6. The mixed lymphocytes culture test was taken at30th after operation. CD4+,CD8+were measuresed by flow cytometry.Result:1. The animal model were built up in all4group rat under the same condition, including weight and operation time without obvious difference (P>0.05), the survival rate is94.66%.(124/131).2. The adipose-derived mesenchymal stem cell and the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells marked with Brdu began to appear after3days of the operation, which nucleus positive of red dye by fluorescence immunohistochemical scattered in liver tissue, and some parts of the positive cells to collect and dense around the blood vessels and abbacy. With the extension of time, gradually formed shape distributings oven.3. Group B, C, D of median survival time is obviously higher than that of the group A (control group)(P<0.05), group D of median survival time was the longest in all group, and were longer than C group,but is no statistical difference compared with group B. 4. Liver function of ALT, AST, TBIL of group A rising sharply, but B,C,D group gradually declined in the postoperative day7th and10th, In the postoperative day14th, animals of group A has completely death, data lost; liver function of group B,C,D further recovery, group B,D recovery degree is better than group C, group D better than B group in some data(ALT,AST)(P<0.05).5. The lever of IL-2,INF-gamma of group A higher than other group significantly (P<0.05), while level of the group B,C,D gradually declined in the postoperative day7th,10th and14th, the lever of group B and D declined more than group C (P<0.05); the lever of IL-10and IL-4of group A was significantly lower than the other group (P<0.05), but group B,C,D gradually raised, group B and D raised more than group C (P<0.05).6. Rejection activity index of group A significantly higher than group B,C,D in the postoperative day7th and10th(p<0.05), group B,D have down trend with time prolonged, rejection index of group C was high than group B and D (p>0.05).7. The mixed lymphocytes culture test:OD value of group B, C and D reduce compared with the positive control group (A) when the stimulate cell come from spleen cells of DA rat. OD value of group B, reduce compared with the positive control group when the stimulate cell come from spleen cells of SD rat. But the OD value of group C,D was still high as the positive control group (p<0.01). It showed that the immune tolerance of recipient was specificity.8. The ratio of CD4+/CD8+of Group A rise progressively after operation,which significantly higher than group B, C and D (P<0.05);the ratio of group B, C and D rise slowly from3th to10th and fall down from14th after operation,but there's no statistical difference.Conclusion:1. Rat liver transplantation high-lever rejection model was established successfully.2. Liver fibrosis and steatosis may appear when adipose-derived mesenchymal stem cells injected by portal vein to intro, which rent differentiation of it to liver cell, the therapy of adipose-derived mesenchymal stem cells only may not enough to protect the body from immune rejection.3. Specificite immune tolerance was successfully established of recipient deal with the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells by portal vein.4. The hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells play a certain role of tolerance establish, without using immunosuppressive agents.
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