| BackgroundLiver transplantation as the only effective treatment for end-stage liver disease is widely accepted, the technology has become more mature and the 5 years-survival rate was more than 70 percent. With the efficient and powerful immunosuppressant widely used, the acute rejection rate has decreased significantly. Consequently, the major factors which affect early survival rate after liver transplantation were more and more concentrated in the liver, kidney, small intestine functional damage. The complications, which include donored liver with no function, acute renal failure and intestinal injury after operation, increased the postoperative mortality obviously.The orthotopic liver transplantation technology is complicated, including three stages: resect, trim and implant donor liver. During the receptor surgery, the portal vein and inferior vena cava need to be block up temporarily,when the blood flow was restore after operation, The tissues of liver, kidney and small intestine have to undergo ischemia-reperfusion injury(IRI). The ischemia-reperfusion injury is the important factor which leads to nonfunction of transplanted liver, acute renal failure and intestinal injury after operation. Apoptosis is the main mode of cell death in the early stage of ischemia-reperfusion injury, many genes involved in the regulation of apoptosis. Bcl-2 and Fas are the important genes related to the apoptosis. Now, more researches focus on the before and after ischemia-reperfusion injury changes and intervention in the liver cell and less in the kidney and small intestine.The relationship between Bcl-2 , Fas gene expression and apoptosis in liver, kidney and small intestine cells after ischemia-reperfusion injury has not yet been reported.Based on the above recognition in present study, we design this experiment to research the apoptosis and Bcl-2 , Fas gene expression in liver, kidney and small intestine cells after liver transplantation, explain the mechanism of the transplanted liver with no function, acute renal failure and intestinal injury after operation , We hope to provide the basis and foundation for research and clinical application to reduce liver transplantation complications.Objective(1) Investigate the technique for performing orthotopic liver transplantation (OLT) in Wistard→Wistard rats stably.(2) Research the changes of apoptosis in the rat liver, kidney and intestinal cells after ischemia-reperfusion injury on liver transplantation , and assess the role of apoptosis in the process.(3)Study the Bcl-2 and Fas gene expression in the liver, kidney and intestinal tissues after ischemia-reperfusion injury on liver transplantation, demonstrate the relations between the apoptosis and Bcl-2, Fas gene expression and explore its possible mechanism.Methods(1) Orthotopic liver transplantation in Wistard→Wistard rats model were performed with the modified "two-cuf "method that described by Kamada and establish control group,The rats were killed at 1h,3h,6h,12h and 24h after operation, liver,kidney and small intestine tissues and blood samples were collected. Plasm levels of alanine aminotransferase (ALT), aspartase aminotransferase(AST), Creatinine(Cr) and blood urea nitrogen (BUN) were measured with automatic biochemical analyser at 1h, 3h, 6h, 12h and 24h after operation. Pathological changes in these tissues were observed at different time.(2)Both the liver transplantation group and the control group, Wistard rats were killed at 1h, 3h, 6h, 12h and 24h after operation. liver, kidney and small intestine tissues and blood samples were collected. the blood serum was obtained and preserved in -70℃refrigerator for inspection. The corresponding pathological specimens of optical microscopy observation were performed. Fas protein expression on apoptosis cells at the different time were detected using irnmunohistochemical staining (sp) method. Flow cell technology and electron microscopy technique were applied at different times in the qualitative and quantitative analysis of the liver, kidney and intestinal cell apoptosis.The Bcl-2 and Fas gene expression in the liver, kidney and small intestine apoptosis cells at the different time were detected by RT-PCR method. ALL data were expressed as means±standard deviation. All calculations were performed by use of the SPSS procedures. P value of <0.05 was considered statisticaly significant.Results(1) The model of orthotopic liver transplantation in Wistard→Wistard rat was established successfully, the operation successful ratio was 90.9 percent.(2) The result of the irnmunohistochemical staining showed that the Fas protein expression in the Experimental group was higher than the control group at the different time, the liver, kidney and small intestinal cells in two groups occurred apoptosis at 1h after operation and reach its peak at 12h. there were significant differences in two groups. (P <0.05)),(3) The result of FCM showed that the apoptotic cells ratio in the liver, kidney and intestinal tissues started to appear at 1h after operation and reach its peak at 12h. The apoptosis in the experimental group was significantly higher than the control group, the difference was statistically significant (P<0.05).(4) The result of RT-PCR showed that the Bcl-2 and Fas gene expressing intensity in the liver, kidney and intestinal tissues started to appear at 1h and reach its peak at 12h after operation. The changes in the experimental group was significantly higher than the control group, the difference was statistically significant (P<0.05).Conclusions(1) Established a stable orthotopic liver transplantation mode in Wistard→Wistard rat through the modified "two-cuf "method that described by Kamada is feasible.(2) Liver, kidney and small intestinal tissues can be caused injury by Ischemia and reperfusion after liver transplantation, especially in the liver and kidney tissues.(3) Liver, kidney and small intestinal cell can be caused apoptosis by Ischemia and reperfusion injury on liver transplantation, which started to increase at 1h and reachits the peak at 12h after operation. The liver cell apoptosis is the highest, follow bykidney, small intestine is the lowest.(4) Liver, kidney and small intestinal Bcl-2 and Fas gene expression were increasedafter the ischemia-reperfusion injury on liver transplantation, which started to increaseat 1h and reach its peak at 12h after operation. Bcl-2 gene expression increased lessthan the rate of Fas gene. Two genes expression and the cell apoptosis changes is thesame in the law. |
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