| Objective To study the mechanism of cholestasis induced by graft warm ischemia following liver transplantation.Methods The model of orthotopic liver transplantation with arterial reconstruction using inbred rats was established under microscopy. The rats were divided into four groups(n=24/group) including the control group and 3 liver transplantation groups in which warm ischemia time was respectively 0, 15, 30 min(W0,W15,W30). Thirty-day survival rates were compared among transplantation groups (n=6/group).To evaluate histology and hepatic function, 6 rats in each group were sacrificed on 3, 7, 14 and 30 days after surgery respectively. The expression of cytokeratin8(CK8), cytokeratin18(CK18), Na~+/taurocholate cotransporter(NTCP) and bile salt export pump (BSEP) mRNAs was analyzed via Real-time reverse transcription-polymerase chain reaction (Real-time RT-PCR) technique. Immunohistochemistry was performed for CK8, CK18, Ki-67and BSEP. Indirect immunofluorescent staining for gap junction connexin-32 (Cx-32) was analyzed by the confocal laser scanning microscopy imaging.Results 1. The long-term survival (longer than 30 days) of WO, W15 and W30 was 100.0% (6/6) ,83.3% (5/6), 66.7% (4/6) respectively.2. Histological studies showed that longer the warm ischemia time, increased in restoration time of the liver as well as aggravated liver parenchyma injury. 3. Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) in all groups showed no marked difference on 3, 7, 14 and 30 days after surgery. 4.Serum alkaline phosphatase (ALP) level increased with the warm ischemia time of the graft and peaked on day 14 postoperatively. The correlation between the warm ischemia time of the graft and serum ALP level was observed. 5. The proportion of Ki67-labeled hepatocytes increased with the warm ischemia time of the graft,, and returned normal on day 14 postoperatively. But the proportion of Ki67-labeled cholangiocytes was also higher after liver transplantation but didn't restored even on 30 days after surgery. 6. The mRNA expression of CK8 and CK18 as well as the protein expression of CK8 and CK18 in hepatocytes decreased with the warm ischemia time of the graft, and returned normal on day 14 postoperatively. 7. The protein expression of CK8 and CK18 in cholangiocytes decreased with the warm ischemia time of the graft and returned normal on 7 days after surgery. 8. The protein expression of Cx-32 was lower with the increased warm ischemia time of the graft andreturned normal on day 14 postoperatively. 9. In all the groups, the mRNA expression of BSEP and NTCP showed no significant difference, as well as the protein expression of BSEP on 3, 7, 14 and 30 days aftersurgery respectively.Conclusions 1. Orthotopic liver transplantation with arterial reconstruction on inbred may be suitable for research on cholestasis induced by warm ischemia of the graft after liver transplantation. 2. These results indicate that rat liver graft could be safely subject to warm ischemia within 30 minutes and will have long-term survival. 3. The main site of warm ischemia of the graft is the hepatocytes. The injury of hepatocyte aggravates with warm ischemia time of the graft, restoration of the function was faster than restoration of the morphology. 4. Cholestasis was evident after liver transplantation at early phase and aggravated with increased warm ischemia time of the graft. Biochemial signs of Cholestasis restored slower than markers of hepatocyte injury. 4. With the increased warm ischemia time of the graft, hepatocytes and cholangiocytes proliferation was higher, but the later restored slowly than the former. 5. CK8, CK18 and Cx-32 took part in early cholestasis after liver transplantation. 6. NTCP and BSEP didn't join in cholestasis early cholestasis after liver transplantation. |
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