Experimental Study On Effects Of Heparin-Binding Epidermal Growth Factor-like Growth Factor On Liver Regeneration After Partial Transplantation In Rats

Part IEstablishment of Partial Liver Transplantation Model in Rats and the Primary Study of Graft RegenerationObjective: To establish the model of partial orthotopic liver transplantation,and then to investigate liver regeneration after partial liver transplantation inrats.Methods: 1. Operational technique was trained in Wistar rats. The partial(60%) orthotopic liver transplanted model was established in Wistar rats withmodified two-cuff technique. The rats were divided into two groups: GroupA receiving 40% liver resection, group B receiving partial livertransplantation. The rats in group A and group B were killed on day 1, 3, and7 after operation. The hepatic graft/body weight ratio, Proliferating CellNuclear Antigen Labeling index (PCNALI), ALT, TBIL, ALB of serum weremeasured.Results: 1. Partial (60%) orthotopic liver transplanted model can beestablished successfuly, and the successfuly rate of model was more than 90percent. 2. Ratio of hepatic graft/body weight: The ratio of hepatic graft/body weight increased to a significantly greater degree in group A than in groupB at postoperative day1, 3 and 7. The rates of liver regeneration reach peak in group A at postoperative day 1, and in group B at day 3. The ideal liver/body weight ratio was restored in group A and B. 3. Biochemical assessment: The serum levels of ALT and TBIL increased on postoperative day 1 in group A and B, but the serum ALT and TBIL in group A were significantly highter than in group B. They had retured to the normal level by postoperative day 7 in both groups. The serum ALB decreasd on postoperative day 1 in both groups, and the serum ALB rentured to normal in group A, but still lower in group B by postoperative day 7. 4.Immunohistochemical detection of PCNA: On postoperation day 1, the group A rats had a significantly larger number of PCNALI than the group B. The PCNALI peaked on postoperation day 1 in the group A, and on day 3 in the group B. The PCNALI of both groups had significantly decreased by postoperative day 7.Conclusions: 1. Partial (60%) orthotopic liver transplanted model can be established successufuly, which can be utilized as a better model to study the mechanism of liver regeneration after patial liver transplantation. 2. Partial (60%) orthotopic liver transplanted can elicit full liver regeneration. 3. The ability of liver regeneration was weaker and the peak of liver regeneration was delayed after partial liver transplantation compared with hepatectomy in rats. Part IIEffects and mechanisms of heparin-binding Epidermalgrowth factor-like growth factor on liver regenerationafter partial transplantation in ratsObjective: To investigate the effects and mechanisms of heparin-binding EGF-like growth factor (HB-EGF) on liver regeneration after partial liver transplantation in rats.Methods: The partial orthotopic liver transplanted model was established in Wistar rats with modified two-cuff technique. 75 recipients were randomly divided into three groups. In recipients, immediate after operation, 0.9% NaCl (group A), 0.1 mg/kg HGF (group B) or 0.1 mg/kg HB-EGF (group C) were administered, respectively, until the rats were killed on day1, 2, 3, 5 and 7 After partial (60 %) liver transplantation. The hepatic graft/body weight ratio were measured. The pathological changes of graft liver tissue were observed. ALT, TBIL, ALB of serum were detected. The expression of PCNA was detected by using immunohistochemistry assay. The expressions of LI-6mRNA and cyclinD1mRNA in liver tissue were examined by Real-Time fluorescent quantitation RT-PCR. Cell cycle, DNA ploidy and apoptosis of liver tissue were evaluated by flow cytometry.Results: 1.Duration of operation was not significantly difference between 3 groups. 2. Ratio of hepatic graft/body weight: The ratio of hepatic graft/body weight increased to a significantly greater degree in group C than in groupA and group B and peaked on postoperative day1. The rates of liver regeneration reach peak in group B at postoperative day 2, and in group A at postoperative day 3. The ibeal liver/body weight ratio was restored in all three groups. 3. Biochemical assessment: The serum levels of ALT and TBIL increased obviously on postoperative day 1 in all three groups, and the serum ALT and TBIL in group A were significantly highter than in group B and C. After postoperative day 1, the serum level of ALT and TBIL had decreased step by step, and retured to the normal level by postoperative day 7 in all groups. The serum ALB decreasd on postoperative day 1 in all three groups, after that the serum ALB increased gradually, and rentured to normal in group B and C but still lower in group A by postoperative day 7. 4. Histology observation: The abundance of fat drops were observed in hepatocytoplasms under light microscopy with HE stain on postoperative day 1 in three groups, especially in group A. The phenomenon of hepatocy swell and balloon-like degeneration Were partially. Cell nuclear mitosis was found in three groups, and more in group C and B on postoperative day 2, 3. The number of liver cell nuclear mitosis in group B and C was significantly higher than that of group A. Heartly status of Liver cell was observed in group B and C after postoperative day 1, and in group A until postoperative day 3. 5. Immunohistochemical detection of PCNA: The PCNA index reached peak at postoperative day 1 in group C, at day 2 in group B, and at day 3 in group A. The PCNA index decreased gradually after peak point in three groups and reached lowest point at postoperative day 7. 6. Liver tissue mRNA expression of IL-6 and cyclin D1 by RT-PCR: 1) mRNA expression of IL-6: Liver tissue mRNA expression of IL-6 reached peak on postoperative day 1, then significantly decreased on postoperative day 2 and 3 in all three groups. There were no significantly differences of mRNA expression of IL-6 among three groups at any time points. 2) mRNA expression of cyclin D1: Liver tissue mRNA expression of cyclin D1 reached peak and significantly highter than group A or group B on postoperative day 1 in group C. On postoperative day 2, liver tissue mRNA expression of cyclin D1 peaked in group B, and highter than other groups. Liver tissue mRNA expression of cyclin D1 peaked in group B, and highter than other groups on postoperative day 3. 7. DNA ploidy and apoptosis of liver tissue were evaluated by flow cytometry: 1) The proliferative index (PI): On postoperative day 1, PI was 13% in group A, 30.6% in group B, and 60.7% in group C, which was highest level and significantly higher than other groups. PI reached peak at postoperative day 2 in group B, and day 3 in group A. 2) Apoptosis index (AI): peak of AI was at postoperative day 1 in all three groups, and decreasd gradually after this time point. There was no differene of AI among three groups. On postoperative day 2, AI in three groups decreasd remarkable than postoperative day 1, and AI rats liver in group A was significantly lower than those in other groups. On postoperative day 5, AI of group C was significantly higher than those of group A and group B. AI of group A at postoperative day 7 was higher than that at postoperative day 5, and significantly higher than those of group B and group C.Conclusions: 1. Administering HB-EGF after partial liver transplantation augments significantly the regeneration and functional recovery of partial liver grafts and reduces hepatocyte injury. 2. Administering HGF after partial liver transplantation can promotes hepatic growth and regeneration and protecting hepatocyte from injury. 3. HB-EGF is more potent than HGF in accelerating G₁/S transition of hepatocyte.