Study Of Liver Co-transplantation With Sertoli Cells In Rats

Objective ①To simplify and improve the method of aparting the Sertoli cells from testicle of rat in order to decrease the cost of separating the Sertoli cells and get cells more pure. ②To improve the method of establishing animal model and acute repulsion animal model of orthotopic liver transplantation in rats in order to find a way to reduce the complication post surgery increase the survival rate after liver transplantation.③ With different ways transferring the culture Sertoli cells into acute repulsion animal model of liver transplantation in rats separately to evaluate the function of immunity protect of Sertoli cells post liver transplantation.Methods ①Seperation and culture of Sertoli cells :Without the process of scissoring testicle into pieces , DNAS digestion and culture under the temperature of 39℃ we grind the testicle tissue of infancy rat on stainless steel gauze to make cell suspension. And after 2 steps of digestion we culture the cells under the temperature of 37 ℃. ②Establishing the animal model of liver transplantation and the acute repulsion animal model of livertransplantation in rats: Applying "Kamada's Cuff technique" to establish the orthotopic liver transplantation animal model of Wistar→Wistar , SD→SD, Wistar→SD, SD→ Wistar in rats . Comparing the different animal models to find the most severe acute repulsion animal model, we modify the way of presurgical medication methods of anaesthesia and surgical technique.③ Co-transplantation of liver with Sertoli cells: We applying for three ways of the co-transplantaton . A:Injection the Sertoli cell suspension into celiac cavity of liver transplantation animal models(n=14). B:Injection Sertoli cell suspension intravenously into liver transplantation animal models(n=14). C: Injection the Sertoli cell suspension into portal vein of donor's(n=14) prior the surgery of liver taking out. We take another 14 cases of acute repulsion animal model of Wistar→SD as control group. Comparing the function of !iver, pathological change and signs post surgery among different groups. ④Injection the Sertoli cell suspension into portal vein of donor's: In order to embed Sertoli cell in donor's liver we blocked the portal vein, suprahepatic inferior vena cave 15 mins after injection.The donor's liver function of prior and post the surgery of blocking vein and 24 hours and 7days after the vein blocked are assayed separately.⑤By using the immunohistochemistry and apoptosis assay technique to analyse the function of Sertoli cell to strive the acute repulsion of liver transplantation. ⑥The data were analysed with SPSS 12.0 by ANOVA and t-test. The criterion for significance is P<0.05. Results ①The modified method of separating Sertoli cells from testicle of rats guaranteed a more pure, high survival rate and production and normal function of Sertoli cells.It has an advantage of low cost and saving time. ② The long-time survival rate achieved more than 90% with almost norejection reaction in liver transplantation animal model of Wistar→Wistar or SD→SD we established. The animal model of Wistar→SD showed the most severe reaction of acute repulsion.③13rats from the group(n=14) of animal model of Wistar→SD with no Sertoli cells co-transplantation died within 14 days post liver transplantation. The group(n=14) of injection the Sertoli cell suspension into celiac cavity had 5 rats living longer than 14 days.The group(n=14) of injection Sertoli cells suspension intravenously had 8 rats living longer than 14 days. The group(n=14) of injection the Sertoli cell suspension into portal vein of donor's prior the surgery of liver taking out had 7 rats living longer than 14 days. The survival rate in co-transplantation groups are 35.7% %, 57.1%, 50.0% separately and has significant difference ( P<0.05) comparing with the control group (7.1%). Pathology examination of liver shows that the groups of co-transplantation appear more lighter reaction of acute repulsion than the control group.④Blocking the portal vein, suprahepatic inferior vena cave 15 mins showed the rat almost no changes of there liver fanction. ⑤The results of immunohistochemistry and apoptosis assay show that Sertoli cell are still alive with secreting the factor of FasL and around the Sertoli cells there are assembling of lymphocytes with some apoptosis lymphocytes after 14 days of co-transplantation. Conclusion ①Simplified and modified method,we used to apart and cultur the Sertoli cells from testicle of rat has the advantages of more effective; saving more time ; having less trauma to the cells; less cost with better function of cells than the routine method. ②The liver transplantation animal model of Wistar→SD appears a feature of severe and stable reaction of acute repulsion it indicates that could be used as a good animalmodel for the relative study of acute repulsion in liver transplantation. ③ Injection the Sertoli cell suspension into portal vein of donor's could embed the Sertoli cell in the liver with blocking the portal vein, suprahepatic inferior vena cave 15 mins almost without damage to liver function. Though there is light increase of AST, ALT, ALP after 24 hours and 7 days , there is no significant difference (P>0.05) compared with control group. ④Sertoli cell could restrain the reaction of acute rejection in liver transplantation and could induce immune tolerance in donor liver.