The Research On Ischemic-type Biliary Lesions Following Clinical Liver Transplantation

Objective: To detect the risk factors of ITBL following liver transplantation. To establish a method to classify ITBL. Verify risk factors of ITBL through prospective study, observed the relationship of cold preservation bile duct injury and ITBL.To observe the relationship between ITBL occur, prolonged cold ischemia and biliary epithelial apoptosis related gene expression.Methods: A retrospective analysis of 886 cases of liver transplant patients was carried on. According cholangiography and radiological examination, diagnosis of ITBL was determine. And all patients were divided into ITBL and NITBL group. Donor factors, receptor factors, surgical factors, postoperative factors between the two groups were univariate and multivariate analysis. Analysis 124 cases ITBL patients mentioned in the first part. According to the severity of biliary imaging, the level of bilirubin, ITBL severity score were given. ITBL was divided into mild, moderate and severe level according to the score. Patients survival, graft survival and adverse prognostic occurrence among the three groups were analyzed by kaplanmeier. 50 cases of liver transplantation patients were observed prospectively. ITBL diagnosis was determined according to cholangiography and radiological examination. And all patients were divided into ITBL and NITBL group. The risk factors in the conclusions of the first part were univariate and multivariate analysis. Bile duct tissues were collected at the time before and after portal vein reperfusion. The pathology change of optical and electron microscopic in biliary was observed. The collected Bile duct tissue were preserved for quantitative real-time PCR(QRT-PCR), to detect MMP-2, Caspase-3, PARP, Bcl-2, Bcl-xL gene level, and for Western-Blot quantitatively to detect MMP-2, Caspase-3 protein levels. Samples were divided into ITBL and NITBL groups, long and short cold ischemia group, before and after portal vein reperfusion groups. Differential gene and protein expression level analysis were performed between the groups,respectively.Results: Retrospective univariate analysis showed that: cold ischemia time(P <0.001), the amount of intraoperative frozen plasma application(P <0.001), anhepatic time(P <0.001),the amount of intraoperative red blood cell application(P = 0.003) was significant statistical difference between the ITBL and non-ITBL group. Multivariate analysis showed that: cold ischemia time(P <0.001), the amount of fresh frozen plasma(P = 0.012), anhepatic time(P = 0.02) was the independent risk factor of ITBL. Patients survival,graft survival, incidence of adverse outcomes have very significant difference(P <0.001) between the ITBL and NITBL group. There is no significant statistical difference of patients survive between mild, moderate and severe groups; There is very significant difference of graft survival between the mild to moderate group(P = 0.004),mild to severe group( P = 0.002); The incidence of adverse outcomes have significant difference between the mild and moderate group(P = 0.014), the mild and severe group(P = 0.001). Prospectively univariate analysis showed that: cold ischemia time(P = 0.033) shows statistically significant difference between the ITBL and NITBL group. Multivariate analysis showed that: liver cold ischemia time, the amount of intraoperative fresh frozen plasma, anhepatic time was not an independent risk factor for ITBL of. ITBL group suffered more biliary epithelial cell loss, apoptosis and the corresponding electron microscopy performance compared to the non ITBL group.ITBL group compared with the non-ITBL group: Bcl-2(P = 0.0082), Bcl-xL(P = 0.021) gene levels were significantly decreased; PARP(P = 0.037),MMP-2(P = 0.021),Caspase-3(P = 0.023) gene expression levels were significantly increased;protein level of MMP-2(P = 0.048), Caspase-3(P = 0.009) were significantly increased. Prolonged cold ischemia group:Bcl-2(P = 0.011), Bcl-xL(P = 0.017) levels were significantly decreased;PARP(P = 0.018),MMP-2(P = 0.014), Caspase-3(P = 0.019) gene expression levels were significantly increased; protein level of MMP-2(P = 0.046),Caspase-3(P = 0.047) were significantly increased. Compared between groups before and after the portal vein reperfusion, the expression levels of each gene were not significantly different. Conclusion: Graft cold ischemia time, amount of fresh frozen plasma, anhepatic time are the risk factors for ITBL occurrence. ITBL decreased patient survival, graft survival, increase the incidence of poor prognosis. The method to classify ITBL according to the severity of biliary imaging, the level of bilirubin have a good prognosis predictive accuracy. The cold ischemia time is a risk factor ITBL occur, ITBL patients had more severe cold preservation biliary duct epithelial injury than NITBL. ITBL occurrence have obvious relevance with apoptosis-related gene level in biliary tract tissue. Prolonged cold ischemia time have obvious relevance with apoptosis-related gene level in biliary tract tissue. Activation of apoptosis system during the graft cold ischemia period may be one of the mechanisms of occurrence of postoperative ITBL.