| Since 1967, Thomas E. Starzl performed the first liver transplantation (LTx) of human being successfully. After nearly 40 years's development, liver transplantation has become the best therapy for the end-stage liver disease. With the development of LTx, postoperative long-term survival of patients is increasing. However, the biliary complication rate of post-LTx still hovers at 5.8 %~24.5%. The case-fatality rate associated with biliary complications is about 6.0 %~12.5%. Therefore, it is important to deal with the biliary complication well to improve long term survival and life quality of LTx patients.However, up to now, the specific etiology of biliary complications after transplantation is not clear. It may be associated with a variety of factors, including ischemic bile duct damage (ischemia-type biliary lesions, ITBL), autoimmune injury and infection damage. Currently ITBL is considered as the main cause of non-surgical graft biliary tree damage. It may involve the following: (1) The direct injury on biliary wall by cold preservation-reperfusion(CPRI); (2)The injury on the bile duct microvascular bed; (3) Graft arterialization delay with heat ischemic injury to the duct; (4) The toxic effects on bile duct epithelial cells by hydrophobic cholatproof.Current research shows that the blood supply of intrahepatic bile duct is mainly from the hepatic artery. The blood supply of intrahepatic bile duct is from the non-axial rete arteriosum, which comes from liver artery branches and accompany with the portal vein branches. The end of hepatic artery and portal vein also are divided into smaller microvascular and associated with the sinusoids.The biliary duct tissue, in particularly the cholangiocytes, is more vulnerable to cold preservation-reperfusion injury(CPRI) and the liver ischemia. The real reasons are not clear. In recent years, the function of microcirculation and vascular endothelial cells in the cold preservation-reperfusion injury has drawn increasing attention. Researches have shown that CPRI may cause the vascular endothelial cell injury of liver, kidney and other organs, and microcirculation obstacles, thereby further increasing the ischemia-reperfusion injury. Combined with clinical cases, cholangiocytes and the PVP are presumed to act the critical role of the microcirculation ischemia pathophysiological process in CPRI. It may be the important reason for the bile duct serious injury and ITBL after long-time cold preservation of donor liver.This study will base on the model of rat liver transplantation and microcirculation ischemia of PVP:â‘ Estestablishing the animal model of PVP ischemia around the microcirculation after liver transplantation;â‘¡Observing the effect of intrahepatic PVP microcirculation ischemia on cholangiocytes;â‘¢Discussing the role of exogenous r-VEGF165 in exploring the relationship between microcirculation dysfunction and transplanted liver intrahepatic biliary epithelial cells in CPRI, and if r-VEGF165 can protect intrahepatic duct and PVP when suffering ischemia. This study aims to further clarify the machanism of transplanted liver cholangiocytes in CPRI, and provide a theoretical reference for preventing the occurrence of ITBL after liver transplantation in clinic. Methods and Results:1. The Sprague-Dawley(SD) rats were performed liver transplantation experiments, and reconstruct hepatic artery with the method of modified Gao's rat liver transplantation. We divided each group into 3 experimental groups randomly according to different interventions factors. We performed second surgery at 3d after the first performing. Group A: hepatic artery ligation (HAL), its subgroups including A1: UW CP 2h, A2: UW CP 8h, and A3: UW CP 16h; Group B: Hepatic Artery microsphere injection and hepatic artery ligation (MS+HAL), also has three subgroups; Group C: control (SO) group. Six rats were sacrificed each time at 3d and 7d respectively, and the liver specimens and blood samples were collected. We selected CP8 group to have histopathologic observation and evaluation. The histological changes of intrahepatic bile duct were more severe in MS+HAL and HAL groups than in SO group. The number of microspheres in implanted liver portal areas was increased more in MS+HAL group than in SO group at the same time point. HAL group was between those two. There were significant difference between MS+HAL group and SO group (P <0.05)).2 On the basis of the first part, we performed immunohistochemical and RT-PCR. The immunohistochemical showed that the protein of PCNA, CK-19, F-â…§-Ag, VEGFA, VEGFB. VEGFC, VEGFR-2 and VEGFR-3 was expressed in cholangiocytes, the strongest expression was in CP2h, CP16h was the weakest, and 8h was in between. Meanwhile, the protein was expressed higher in 7d than in 3d. There were only a few non-significant differences in the same group at different time or in the different group at the same time. There was no protein expression of VEGFR-1. We also selected CP2 group for RT-PCR revealing the cholangiocytes expressed mRNA of VEGF and its receptor in all subgroups. Identity ratio showed that only a few non-significant differences were found in the same group at different time or in the different group at the same time and it was higher in 7 days than in 3 days.3. We performed second surgery at the 3d after the first performing in P8 group and devided it into 3 subgroups. Group A: hepatic artery ligation (HAL) +r-VEGF165 2ml (2.5ng/ml) intraperitoneal injection(HALV) ; Group B : Hepatic Artery microsphere injection + hepatic artery ligation (MS+HAL)+ r-VEGF165 2ml intraperitoneal injection(MSHALV); Group C : control (SO) group(HAL and MSHAL). Six.rats were sacrificed each time at 3d and 7d respectively, and the liver specimens were collected. We selected CP8 group to have histopathologic observation and evaluation. The histological changes of intrahepatic bile duct were less in HALV or MSHALV groups than in HAL and MSHAL groups. The number of microspheres in implanted liver portal areas was decreased a lot in HALV group than in SO group at the same time point. There were significant difference between the two groups(P <0.05)). Immunohistochemical showed that the protein of PCNA, CK-19 , F-â…§-Ag, VEGFA, VEGFB. VEGFC, VEGFR-2 and VEGFR-3 was expressed in cholangiocytes, the strongest expression was in HALV, MSHAL was the weakest, and others were between the above two. Meanwhile, the protein expression at 7d is higher than that in 3d. There were only a few non-significant differences in the same group at different time or in the different group at the same time. There were no protein expression of VEGFR-1. We also selected CP2 group for RT-PCR revealed that cholangiocytes expressed mRNA of VEGF and its receptor in all subgroups. Identity ratio showed that only a few non-significant differences were found in the same group at different time or in the different group at the same time and it was higher in 7 days than in 3 days.Conclusion:1. We established the animal model of PVP microcirculation ischemia after liver transplantation successfully.It can be used in studying peripheral vascular plexus duct microcirculation ischemia on cholangiocytes after liver transplantation;2. PVP microcirculation ischemia may stimulate cholangiocytes secrete and express the protein of VEGFA, VEGFB. VEGFC and their receptor VEGFR-2. VEGFR-3, which could improve ischemia of duct peripheral vascular plexus microcirculation and induce the compensatory hyperplasia of duct peripheral vascular plexus microcirculation , and indirectly induce the repairation of cholangiocytes;3. Exogenous r-VEGF165 could improve the circulation of PVP microcirculation, therefore indirectly prevent the bile duct injury.
|
PDF Full Text Request