Effect And Mechanism Of RhGH On Cholestasis And Liver Function At Early Stage After Transplantation

PARTⅠESTABLISHMENT OF THE RAT LIVER TRANSPLANTATION MODELObjectiveTo study the operation technique of rat's orthotopic liver transplantation(OLT) and to provide a reliable model for the study of OLT.MethodsThe models of the Wistar rat were established with modified Kamada's two cuff technique.We monitored the survival rate complications of operated rat.ResultsAbout 220 cases of rat orthotopic and liver transplantation operations were performed in total.In 40 cases of formal operations,the operation time of donor was(22.3±7)min,cuff preparation time of the whole graft group was(15.6±4)min,the operation time of recipient was(50.6±11.2)min,anhepatic phase was(20.6±2.5)min.The successful rate and 1 week survival rate were 100%and 92.5%respectively.We successfully establish a stable rat liver transplantation model.ConclusionsIf you want to establish a stable rat liver transplantation model,you must master enough microsurgery skills and an intimate knowledge of abdomen anatomy of rat. Moreover,you have to repeat training in OLT for a phase.The sophisticated microsurgical technique and the delicate surgical manipulation is the key to successfully perform rat orthotopic liver transplantation. PARTⅡEFFECT AND MECHANISM OF rhGH ON CHOLESTASIS AND LIVER FUNCTION AT EARLY STAGE AFTER TRANSPLANTATIONObjectiveOur experiments were designed to study the influence and regulatory mechanisms of rhGH on cholestasis and liver function after liver transplantation in rats.Materials and MethodsOrthotopic liver transplantation(OLT) was performed in male Wistar rats using the cuff technique as described by Kamada with modifications.Rats were divided into three groups randomly:SO group(n=18,sham operation group),OLT group(n=18,liver transplantation control group),OLT+rhGH group(n=18,rhGH administrated group in liver transplantation),Each group was further divided into 3 subgroups respectively according to different stage after operation.Three time points were predetermined as 1d,3d,7d postoperation.Six animals were used per time-point.Rats were sacrificed at each time-point and the liver specimens and blood samples were collected.The liver function was evaluated by serum ALP,GGT,ALT,TBIL levels.Levels of TNFα,IL-1βwere measured by ELISA.Serum GH by radioimmunoassay.The GH receptor and IGF-1 receptor binding in grafts were analyzed by radioligend binding assay(RBA).Levels of ATP in hepatic tissues were examined by high performance liquid chromatography(HPLC).The change of Na~+/K~+ATPase was measured by chromatometry.Finally,Mrp2 of liver tissue was determined by immunofluorescent microscopy and RT-PCR.Meanwhile,expression of Mrp2mRNA correlatively analyzed with TNFαand TBIL in the serum.Morphometry was observed under light microscope and TEM.Results1.Biochemical data showed that the levels of serum ALP,TBIL,GGT,ALT and TNFαwere significantly lower in OLT+rhGH group than that in OLT group(P＜0.05), while the level of IL-1βbetween the two groups has no significant difference.2.At 1,3 days after OLT,the levels of ATP and the activity of Na~+/K~+ ATPase were significantly lower in OLT group than that in SO group(P＜0.05).Treatment with GH,the levels of ATP and the activity of Na~+/K~+ ATPase significantly increased.3.At 1d after operation,the level of serum GH in group OLT was not different compared with that in group OLT+rhGH and SO.while At 3,7 days after OLT,the level of serum GH was significantly lower in OLT group than that in SO and OLT+rhGH group(P＜0.05).In OLT group,the liver grafts showed a significant decrease in GH receptor and IGF-1 receptor number on postoperative day 7(P＜0.05).After the rhGH treatment, GH receptor markedly recovered on postoperative day 7 while IGF-1R did on postoperative day 3 and 7(P＜0.05).4.At 1d after operation,the expression of Mrp2 in OLT group was so weak that could hardly be detected under fluorescence microscope,while obvious fluorescence could be detected from the seventh day postoperatively,showing significant discrepancies when compared with SO group.As duration elongated,both the intensity and range of specific fluorescence grew,moderate level of fluorescence was maintained.Compared with OLT rats at the same timepoints,the expression of Mrp2 in rhGH-treated rats was much stronger.Expression of Mrp2mRNA correlated negatively with TBIL and TNFα..5.The histological changes were more severe in OLT group than that in OLT+rhGH group.Conclusions1.Cholestasis was evident after liver transplantation at early phase.Restoration of the function was faster than restoration of morphology.Exogenous GH treatment can protect ischemic/reperfusion injury in rat liver,and facilitate recovery of function of liver graft.2.The level of ATP and Na~+/K~+ ATPase in early time of cryopreservationreperfusion in donor liver are related to the graft function.The changes of Na~+/K~+ ATPase in graft indicated that liver transplantation might lead to energy metabolism disturbance of the membrane of liver cell,which might be responsible for the metabolism disorder of cholestasis.3.Changes of GH-IGF-1 axis appeared,expression of GHR and IGF-1R trended to descend within the first 7 days postoperatively.Injury from warm ischemia caused delayed recovery of GH-IGF-1 axis,and transient GH resistance was present.Both GHR and IGF-1R decreased and resulted in a relative deficiency of GH.The delayed recovery of function of liver graft was implicated to the changes of GH-IGF-1 axis.After OLT, exogenous GH treatment facilitated recovery of GH-IGF-1 axis,and recovery of function of liver graft was promoted by postoperative rhGH treatment.4.The level of Mrp2 in the liver tissue was upregular by rhGH and was significantly.Liver transplantation might lead to down-regulation and abnormal localization of Mrp2 in liver cell,which might be responsible for the cholestasis after rat liver transplantation,rhGH can protect the liver from the injury of intrahepatic cholestasis.The promotion of bilirubin transport by rhGH can upregulate the expression of Mrp2 directly.On the other hand,The application of rhGH might limit exaggerated hepatic production of TNFαto upregulate the expression of Mrp2.