Experimental Study On The Effect Of Primary Rat Hepatocytes Intrasplenic Transplantation Combined With CsA In Rats With Acute Liver Failure

Objective To investigate the effect of primary rat hepatocytes intrasplenic transplantation with cyclosporine A(CsA) on acute liver failure(ALF) induced by thioacetamide(TAA),and to evaluate the influence of CsA on HCT,providing a clinical application ofhepatocytes transplantation(HCT) theoretically.Methods1.Forty-eight male SD rats were randomly divided into 4 groups.TAA solution was injected to each group via the abdominal cavity with dosages of 300,350,400, 450mg·kg^-1·d^-1 respectively at the same time for two days.Clinical manifestation,the number of deceased rats,hepatic biochemical function,prothrombin time(PT) and pathological changes were observed and recorded in each group at different time to determine the optimal dosage and time for preparation of the ALF model.2.The improved simple perfusion system was used to isolate rat hepatocytes by a two-step perfusion with collagenase in situ.Hepatocytes were separated from the dissociative cells by low speed centrifugation(500rpm,3min,3times ),and discontinous gradient centrifugation method was applied to purify the hepatocytes. Cell viability was required to be 85%or greater to be considered acceptable for HCT.3.Eight of sixty-eight SD male rats were chosen as hepatocyte donors.Of the sixty SD rats with ALF induced by TAA after intoxication,eleven rats died in 24h. Surviving rats were randomly divided into 3 groups:Groupâ… 17 rats and other two groups 16 rats respectively.Groupâ… received 1ml of approximately 2×10⁷ hepatocytes suspension and CsA 10 mg·kg^-1·d^-1,through intrasplenic injection and intramuscular injection.Groupâ…¡only received 1ml of approximately 2×10⁷ hepatocytes suspension.Groupâ…¢as control received 1 ml of normal saline.The survival rate,hepatic biochemical function and pathological changes of transplanted hepatocytes and liver of all rats were observed.Results1.According to the experimental observations,the dosages of 350mg·kg^-1 and 24 hours after intoxication were determined as the optimal one and time for inducing ALF model.2.The viability of hepatocytes obtained by the two-step perfusion with collagenase in situ was 92.63±2.50%after applying a discontinuous gradient centrifugation method.3.Comparision of one-week survival rates with each other group,groupâ… and groupâ…¡were higher than that of groupâ…¢(64.7%vs 12.5%,P = 0.003＜α;and 56.3%vs 12.5%,P = 0.012＜α,respectively ).Survival rates of the other two groups were similar.On the 1^st day after operation,there were no difference in serum biochemical indicators among three groups(P＞0.05).On the 3^rd or 4^th day after operation,each group rats began to regain their weight loss,and weight of groupâ… and groupâ…¡significantly increased compared to that of groupâ…¢(P＜0.05), whereas there was no significant difference in weight between groupâ… and groupâ…¡(P＞0.05).Serum ALT and AST for groupâ… andâ…¡experienced obvious improvement compared to groupâ…¢,especially groupâ… .Serum TBIL for groupâ… andâ…¡was improved statistically,compared to groupâ…¢(P＜0.01,P＜0.05, respectively),but the difference was not statistically significant between groupâ… andâ…¡.There was significant difference on serum PAB between groupâ… andâ…¢(P＜0.05),whereas that of other groups was similar.On the 7^th day after operation, serum PAB for groupâ… andâ…¡was improved remarkably,compared to groupâ…¢(P＜0.05),however there was no statistical significance between groupâ… andâ…¡. Liver function indicators returned to normal except for PAB.On the 7^th,14^th day after operation,the hepatic pathological state of groupâ… andâ…¡experienced remarkable improvment,especially groupâ… .There was a minimal inflammatory reaction around liver cells transplanted into recipients and prolonged survival time in groupâ… , compared to groupâ…¡.On the 14^th day,surviving hepatocytes were still seen in groupâ… ,while complete hepatocytes were very difficult to see in groupâ…¡on the 14^th day.Conclusion1.Abdominal injection of TAA with dosage of 350mg·kg^-1 and 24 hours postinjection is the optimal dosage and time to induce ALF on rat model in this experiment.2.The viability and purification of hepatocytes obtained by modified two-step collagenase perfusion method in situ were raised remarkably after percoll discontinous density gradient centrifugation meet the need of HCT.3.The ameliorative effect of intrasplenic hepatocytes transplantation on rats with ALF was significant,improving the survival rate of rats with ALF induced by TAA. Although the survival rate of the combined treatment group could not to be improved further,it could remarkably ameliorate hepatic function and pathological state and prolong survival time of liver cells transplanted into the recipient...