| BackgroundLiver transplantation as the only effective treatment for end-stage liver disease has been widely accepted. Although after many years of development, the peri operative mortality rate has dropped to about 5%,10 years survival rate reached more than 80%, but donor liver which was caused by ischemia reperfusion injury in vitro is still one of the problems need to be solved. It was found that the liver ischemia-reperfusion injury is one of the main causes of primary liver failure.In recent years, people began to search for effective ways to reduce ischemic reperfusion injury, but it still did not completely solve this problem.Some scholars put forward the measures to block the portal vein in liver resection and reduce the ischemia reperfusion injury. Since then, researchers in the method of myocardial ischemia reperfusion injury in the ischemic postprocessing to reduce myocardial injury. With the development of molecular biology, it has been paid more attention to the improvement of the target gene in vivo or in the body to relieve the ischemia-reperfusion injury. Such as recombination of gene of interleukin-1(IL-1), interleukin-13(IL-13), Heme oxygenase-1(HO-1) expressed in vivo.In recent years, which is called transplantation of protective gene, has become a hot research topic. It is the rate limiting enzyme of heme metabolism, which expressed considerable amount and widely distributed in vivo mammalian. And Kupffer cells were inhibited to secrete inflammatory mediators in the liver. HO-1 is highly inducible, which highly heat, hypoxia, toxin and inflammation can be induced the expression to protect the liver. In previous studies, HO-1 has been found to reduce the ischemia-reperfusion injury of transplanted liver, but its mechanism is still unclear. It is very important for peribiliary vascular plexus after liver transplantation. It is important foundation of intrahepatic biliary blood supply sources and biliary tract structure and function recovery after liver transplantation. It has been reported that HO-1 can improve the microcirculation of the biliary tract, but the effect and specific ways remains problems.ObjectivesAccording to the protective effect of HO-1 on liver ischemia-reperfusion injury and the characteristics of bile duct ischemia-reperfusion injury in liver transplantation, By reformed "two cuff method", rat orthotopic liver transplantation model was established and the induced and suppressed HO-1 gene adenovirus were constructed. To change the expression of HO-1 gene in rat by adenovirus transfection, and then to analyse the ischemia reperfusion injury of liver transplantation, ways of action and mechanism of HO-1 transplantation on regeneration of peripheral vascular plexus.Methods:1.160 rats were divided randomly following 5 groups, per 32, donor induce (donor was injected HO-1 induced adv and recipient was injected HO-lsuppresed adv, induce/suppress I/S), donor and recipient induce group (donor and recipient were injected HO-1 induced adv, I/I), recipient induce group(donor was injected HO-lsuppresed adv and recipient was injected HO-1 induced adv, S/I), donor and recipient surppress group (donor and recipient were injected HO-1 suppressed adv, S/S) and control (donor and recipient were injected empty adv, Control C).2. Before surgery 24 hours, each of rat had been injected adenovirus (0.5ml 2* 109) with HO-1 induced or HO-1 suppressed by the rat via tail vein. And then expression of level was tested.3. methods of surgery:200-220g adult rats were selected, reformed "two cuff method" was used to constructed rat orthotopic liver transplantation model.4. serum test:After liver transplantation,1D,3D,7d and 14d, the serum were used to detect the liver function by using the full automatic biochemical analyzer. The serum levels of hepatocyte growth factor (HGF) were detected by ELISA method.5. Morphological changes of liver tissue were observed by light and electron microscope, and Suzuki score of liver tissue pathology was also judged.6. level of expression of hypoxia inducible factor-1 alpha (HIF-1), vascular endothelial growth factor (VEGF), HO-1 was detected by western blot in liver tissue.7.Frozen specimen was used to laser confocal microscopy of cytokeratin 18 (CK-18) and laminin (LN) double immunofluorescence, von Willebrand factor (vWF) immunofluorescence and the number of blood vessels were calculated.8. Values of different parameter were expressed as mean±SD. Statistical analysis were performed using SPSS 17.0 (Inc, Chicago, IL, Unite States) for Windows. Difference of between groups were analyzed by one-way ANOVA analysis of variance or t test, multiple comparisons were analyzed by LSD of post hoc test. P value<0.05 was considered statistically significant. Results1.serum results:Liver function results:after operation 7d,14d, group I/S and I/I, AST, ALT, ALP, GGT were lower than groupS/I, S/S and empty virus group. At the same time empty virus group AST and ALT were lower than group S/I and S/S, the difference was statistically significant (p<0.05).ELISA results:after operation 7d, group I/S HGF (229.43+77.05) pg/ml, group I/I was (248.57+68.48) pg/ml higher than group S/I (131.29+24.40) pg/ml and group S/S (109.12+26.92) pg/ml, the difference was statistically significant (P<0.05).2. Pathological biopsy results:On the pathological morphology, group I/S and I/I to group S/I and S/S, the degree of liver cell edema lighter, less inflammatory cells infiltration, the department of portal area structure is normal. The Suzuki score of group I/S and I/I was lower than group S/I, S/S and empty virus group, and the Suzuki score of empty virus group was lower than group S/I and S/S, and the difference was statistically significant (p<0.05).3. Transmission electron microscopy:There are bile duct epithelial cell structure is normal, microvillis of bile duct in the group I/S and I/I. However, group S/I and S/S, bile duct epithelial cells can be seen lysosomes which have different sizes, endoplasmic reticulum and Golgi expansion.5. Western blot results:After postoperation 24h, expression level of HO-1 VEGF(24KD) in the group I/S I/I increased more than group S/I S/S, there is statistical difference(p<0.05). After postoperation 7d, expression level of HO-1, HIF-la, VEGF(43KD) and VEGF(24KD) increased more in the I/S I/I group, as compared to the group S/I S/S, and there were significant difference by observed (p<0.05).6. Immunofluorescence results:Comparasion with group I/Sand I/I, the group S/I and S/S the structure of the cell was g, the structure of the bile ducts and blood vessels were better, and the number of newborn small vessels was more. The number of blood vessels in the group I/S and I/I was significantly more than that in the group S/I, S/S and empty virus group, and the difference was statistically significant (p<0.05).Conclusion1.The reformed "two cuff technique in rat liver transplantation model ensures that the rat survival rate. It is a kind of stability and strong operability and more in line with the physiological and pathological process clinical liver transplantation model.2.HO-1 overexpression can effectively reduce liver cell edema and inflammatory cell infiltration and accelerate the recovery of liver function, alleviate hepatic ischemia reperfusion injury; conversely, inhibition of HO-1 expression, is not conducive to liver transplantation from ischemia and reperfusion injury recovery.3.HO-1 over expression can induce the expression of HIF-1 and VEGF and increased intrahepatic vascular regeneration, promote recovery of structure and function of the biliary tract, reduce periportal infiltration of inflammatory cells, alleviate liver transplantation biliary ischemia reperfusion injury.4. HO-1 gene plays a major role in liver transplantation in liver transplantation... |
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