Effect Of Donor Apoptotic Lymphocytes On Cytokines In Acute Rejection Model Of Rat Liver Transplantation

Chapter 1. Technology improvement of rat orthotopic liver transplantation and establishment of acute rejection modelObjective: to improve the classical "two-cuff method", build experimental model of rat orthotopic liver transplantation (ROLT), observe the development process and basic pathophysiologic changes of acute rejection following allotransplantation from DA to Lewis rat, and establish the ideal technology platform for the following study in that apoptotic splenic lymphocyte induce transplantation immune toleranceMethods: During preliminary experiment phase, 150 models of ROLT were builded from closed population SD rats to SD rats with the improved "two-cuff method " to make the technology more skilled and improve the stability. Inbred strain rats were randomly divided to two groups: A. Isogene group : ROLT from Lewis to Lewis. B. Allogene group: ROLT from DA to Lewis. Recipients were sacrificed on 3rd d, 5th d, 7th d, and 10th d postoperatively and liver tissues and blood samples were collected. Time during major surgery steps, recipient's general state of health and survival time after operation, histopathological characteristics were observed. Plasm levels of aspartate transaminase (AST) and total bilirubin (TBil) were measured with automatic biochemical analyser. Results: ROLT models were builded after 150 models had been done. Donor operating time was 27.9±1.3min, grafts preparing time was 12.0±1.1min, recipient operating time was 55.4±2.3min with anhepatic period of 17.9±0.9min. One week survival rate of recipients was 93.3%(28/30). Isotransplantation from Lewis to Lewis rat resulted in immune tolerance, and the medial survival time was more than 100 days. It was seen that mild morphologic changes occurred in hepatic tissue. Slight denaturation and vacuolation of hepatocytes were found. Hepatic sinusoid expanded slightly and was filled with few erythrocytes. Few inflammatory cells infiltrated in portal area, and the structure of hepatic lobules remained. On the contrary, recipients after allotransplantation performed jaundice obviously and became thinner and thinner. The medial survival time was 11 days. Hepatic histological examination showed typical acute rejection on 7th d in Williams standard. Lots of vacuoles were seen in the cytoplasm of hepatocytes and more necrotic hepatocytes could be also observed. Hepatic sinusoid expanded obviously and was full of erythrocytes. Many inflammatory cells infiltrated in portal area, and the architecture of liver lobules was destroyed. On 3rd d postoperatively, AST between two groups was not significantly different(P=0.741), and TBil of isogene group was higher than one of allogene group(P=0.004). On 5th d, 7th d and 10th d postoperatively, however, recipients' AST and TBil level in allogene group was sinificantly higher than that in isogene group(P≤0.001).Conclusions: With improved "two-cuff method", high success rate and wonderful stability were chieved in establishing models of ROLT. With the results of recipients general status of health, survival time, hepatic histological examination, and serum liver function, which performed similar as clinical features of acute rejection, a acute rejection experimental model of liver transplantation in rat could be determinded using DA as donor and Lewis as recipient. We could examine the rejective degree on 7th d postoperatively, when recipient performs representative rejection. At the same time, the following study in that apoptotic splenic lymphocytes induce transplantation immune tolerance could be performed with this acute rejection model. Chapter 2. Effect of donor apoptotic lymphocytes on cytokines in acute rejection model of rat liver transplantationObjective: To understand the inhibiting effect of donor apoptotic splenic lymphocyte on acute rejection on the basis of establishment of acute rejection model of rat liver transplantation, and to study the mechanism of immune tolerance induced by apoptotic cell through observing the change of cytokine in liver tissue and peripheral blood of recipients after transplantation.Methods: Donor necrotic splenic lymphocytes were prepared by aqueous bathing in 56℃for 30 minutes. Donor apoptotic splenic lymphocytes were prepared by 2.0Gy irradiating with accelerator linear and cultivating for 8 hours. The cells of two groups were checked by the method of Annexin V plus PI with flow cytometer. Allotransplantation from DA to Lewis rat was performed with improved two-cuff method, and recipients were randomly divided into three groups:l. Control group, in which recipients received no treatment before transplantation; 2. Necrosis group, in which recipients were transfused donor necrotic splenic lymphocytes by vena dorsalis penis 7th day before transplantation; 3. Apoptosis group, in which recipients were transfused donor apoptotic splenic lymphocytes by vena dorsalis penis 7th day before transplantation.The following examinations were performed:1. 4 recipients of every group were feed for recording survival time, and the deadline is 100d after transplantation.2. Recipients were sacrificed on 7th d postoperatively and liver tissues and blood samples were collected. Histopathological characteristics were observed. Plasm levels of aspartate transaminase (AST) and total bilirubin (TBil) were measured with automatic biochemical analyser. 3 rats of every group were examined at every timepoint.3. Recipients were sacrificed on 3rd d, 5th d, 7th d, and 10th d postoperatively and liver tissues and blood samples were collected. Time during major surgery steps, recipient's general state of health and survival time after operation, histopathological characteristics were observed. Plasm levels of aspartate transaminase (AST) and total bilirubin (TBil) were measured with automatic biochemical analyser. Tissues were treated by schizolysis in low temperature and centrifuged. Cytokines are assayed by Luminex liquid array(IL-2,IL-4,IL-10,IFN-γ).Results:1. The cells' necrotic rate was 98.561% which were treated by aqueous bathing in 56℃for 30 minutes. The cells' necrotic rate was 50.408% which were treated by 2.0Gy irradiating with accelerator linear and cultivating for 8 hours.2. The survival times of three groups were significant different (F=110.667,P=0.000).The medial survival time of control group is 11 days, and thatof necrosis group is 10 days. There was no significant difference between these two groups. The medial survival time of apoptosis group was over 100 day, more than the former two groups.3. The AST level and TBil level of necrosis group were more than ones of control group(P=0.010, 0.024). On the contrary, the AST level and TBil level of apoptosis group were less than ones of control group(P=0.000).4. Hepatic histological examination of control and necrosis group showed serious rejection on 7th d in Williams standard. Lots of vacuoles were seen in the cytoplasm of hepatocytes and more necrotic hepatocytes could be also observed. Hepatic sinusoid expanded obviously and was full of erythrocytes. Many inflammatory cells infiltrated in portal area, and the architecture of liver lobules was destroyed. On the contrary, the examination of apoptosis group showed mild rejection. liver cell cord and lobula construction were still normal. There were cloudy swelling and punctiform necrosis in among of hepatocytes. Few of inflammatory cells infiltrated in portal area.5. Three groups had significant different effects on the level of IL-2,IL-4,IL-10,IFN-γin liver tissues. The time points after operation systematically affected the level of IL-2,IL-4,IL-10,IFN-γin liver tissues. The IL-2,IFN-γlevel in liver tissues of control group were less than those of necrosis group(P<0.05), but more than apoptosis group(P<0.05). Apoptosis and control group had no significant different effects on level of IL-10 on 3rd d postoperatively. The IL-4,IL-10 level in liver tissues of control group were more than those of necrosis group(P<0.05) and less than apoptosis group(P<0.05) at other time points.6. The control group and necrosis group had no significant different effects on the level of IL-4,IL-10 in serum on 3rd d, 7th d,10th d postoperatively(P>0.05),but the levels of IL—2,IFN—γin serum of necrosis group were more than those of control group(P<0.05). The levels of IL—2,IFN—γin serum of apoptosis group were more than those of the former two groups (P<0.05) . The levels of IL—2,IFN—γin serum of apoptosis group were less than those of the former two groups on 3rd d,7th d,10th d postoperatively (P<0.05) .However, The levels of IL—4,IL—10 in serum of apoptosis group were more than those of control and necrosis group, except of the level of IL—4 on 3rd d.Conclusion:1. The treatment by 2.0Gy irradiating with accelerator linear and cultivating for 8 hours could efficiently induce rat splenic lymphocytes apoptosis.2. The results of hepatic histological examination and serum liver function showed that donor apoptotic splenic lymphocytes pretransplantation 7th day before operation could obviously induce transplantation immune tolerance, on the contrary, the necrotic cells had adverse effect. The survival time indicated that apoptotic cells could induce long-time surviving of allograft.3. The result of cytokine showed that donor donor apoptotic splenic lymphocytes pretransplantation could up-regulate the Th2 cytokines, including IL-4, IL-10 and so on, but inhibited the Th1 cytokines, including IL-2,IFN-γand so on. It also indicated possible relation between immune tolerance induced by apoptotic cells and Th1/Th2 balance.4. Further work needed to be done to indentify some other related immune elements and functions, such as Treg, memory T cell, and B cell, and to understand their significance in the induction of transplantation tolerance.