| Part one Establishment of orthotopic liver transplantation model in tree shrews[Objective]To attempt to establish an allogeneic orthotopic liver transplantation animal model in tree shrews for the first time,which provides a new kind of orthotopic liver transplantation model for clinical and scientific research,and has higher homology with human genotypes and immune system.The animal model of liver transplantation is more economical and practical.[Methods]On the basis of "two-cuff’ technique liver transplantation model reported by Kamada.60 tree shrews were randomly divided into the donors and the recipients for orthotopic liver transplantation.The success rate of operation,the survival rate of tree shrew after operation and histopathological changes were observed to construct a stable orthotopic liver transplantation model for tree shrews.[Results]The success rate of stereotyped surgery was 90%(27/30),The 24-hour survival rate of tree shrews was 76.67%(23/30).The three-day survival rate was 60%(18/30),meanwhile the one-week survival rate was 30%(9/30),and the longest survival time was 11 days.The causes of operation failure were as follows:Anastomotic hemorrhage of superior and inferior hepatic vena cava,excessive anaesthesia and pneumothorax with 1 cases in each.Four cases of death within 24 hours,of which 1 case died of anastomotic hemorrhage and 1 case died of anastomotic thrombosis,two causes of death was unknown.23 cases died after operation for more than 24 hours,the causes were acute rejection in 15 cases(65.22%),biliary obstruction in 1 case,abdominal cavity infection in 2 cases,liver lobe necrosis in 1 case,four causes of death was unknown..[Conclusion]1.The construction of orthotopic liver transplantation model on tree shrew is more difficult than that in rat liver transplantation,and there are more influencing factors.Skilled microsurgical techniques,perfect perioperative management and patient and meticulous operation are the key to successful surgery.2.The tree shrew liver transplantation model is the innovation of this study,and has not been reported yet.The orthotopic liver transplantation model of tree shrews was successfully established by using tree shrews as the first successful orthotopic liver transplantation model.As a new animal model of liver transplantation,the modified"two-cuff" method was established in the orthotopic liver transplantation model of tree shrews,and the model was stable and reliable.,which lays a foundation for the clinical and basic research of the application of the tree orthotopic liver transplantation model to liver transplantation in the future.Part two Study on the migration and expression of CXCL12/CXCR4 gene in lymphocytes of tree shrew in Vitro[Objective]To study the effect of rapamycin on the chemotactic function of peripheral blood lymphocyte chemokine CXCL12(SDF-1)and the receptor CXCR4 expression in tree shrews,and provide experimental evidence for the study of transplantation rejection in vivo.[Methods]Experimental group:rapacymin drug group、DMSO control group and blank group.The effect of rapamycin on the survival of tree shrew lymphocytes was detected by MTS cell proliferation test,The effects of rapamycin on human recombinant CXCL12 induced lymphocyte migration in tree shrews were detected by cell migration experiments and the effect of rapamycin on the expression of CXCR4 gene in lymphocytes of tree shrew was detected by Real-time PCR.[Results]Rapamycin had no significant effect on lymphocyte activity of tree shrews.But it inhibited the migration of lymphocytes in tree shrews.At the same time,rapamycin down-regulated the expression of CXCR4 gene in lymphocytes of tree shrews.[Conclusion]The expression of chemokine CXCL12 and the receptor CXCR4 can affect the migration of lymphocytes,which may provide evidence for the experimental study of acute rejection after liver transplantation in tree shrews.Part three Study on the role and mechanism of CXCL12/CXCR4 expression in acute rejection of tree shrew liver transplantation[Objective]The function of chemokines and their receptors were studied with immunosuppressive agents and chemokine receptor inhibitors on the basis of liver transplantation model.Through the differences in expression,the role and mechanism of chemokine/chemokine receptor(CXCL12/CXCR4)in the regulation of lymphocyte migration in acute rejection of liver transplantation were elucidated.It provides an important basis for the mechanism of acute rejection of liver transplantation,and can provide experimental data and theoretical support for clinically effective treatment and control of acute rejection of grafts and promotion of graft survival.[Methods]1.Establishment of the acute rejection model for liver transplantation in tree shrew,as shown in part one.2.Experimental group:(1)control group,group A(n=25):normal tree shrews were used as the control group;(2)The exclusion group,group B(n=25):allogeneic liver transplantation of tree shrews;(3)group AMDS 100,group C(n=25):Intravenous AMD3100(1 mg/kg/day)in tree shrew allogeneicliver transplantation;(4)Rapacycin(RAPA)inhibition group,group D(n=25).Five tree shrews were randomly sacrificed on the 1st,3rd,5th and 7th days after surgery,and blood,liver and spleen samples were collected.5 tree shrews were left to observe the survival time and draw the survival curve.3.Biochemical measurement:Serum was collected on Id,3d,5d and 7d.after liver transplantation in each group,and the levels of TP,ALT,AST and TB were measured with the blood automatic biochemical analyzer.4.Detection of lymphocyte CXCR4 by flow cytometry:splenic lymphocytes were collected at 1,3,5,7 days after liver transplantation for isolation,culture and flow cytometry.5.Histological examination of transplanted liver:Immunohistochemical staining(IHC)was used to detect and analyze the expression of CXCL12.Pathological examination of liver tissue HE staining and the degree of rejection was determined according to the Banff scoring system.6.Enzyme-linked immunosorbent assay(ELISA)was used to detect the amount of CXCL12(SDF1)protein in plasma.7.Western blot analysis:the expression of CXCR4 protein was detected in each group.[Results]1.The median survival time of group C and group D was significantly longer than that of group B,and the difference was significant(P<0.05).All groups were compared with group A(P<0.05).2.Biochemical examination of tree shrews in each group after liver transplantation:The correlation indexes of 1d,3d,5d and 7d after liver transplantation in each group were compared.Postoperative serum TP was significantly lower in group B,C and D than that in group A(P<0.05).On the 1st day,the TP began to decrease in group B,C,D,there were no statistical differences in other groups(P>0.05).Group B showed a continuous decrease,while group C and D began to recover with the extension of time on the 5d and 7d days after surgery,showing an upward trend,and the difference was significant(P<0.05)when compared with group B at the 5d and 7d time points of group C and D.TP in group D increased better than group C on the 3rd day after operation,but there was no difference in statistics between the two groups(P>0.05).The serum levels of TB,ALT,AST in group B,C and D increased sharply.TB,ALT,AST,in the same period of 1,3,5,7 days after operation were more higher in group B,C,and D than that of in group A(P<0.05).while in group C and D,5 days and 7 days after operation,the liver function began to recover with a slight upward trend,but it was relatively flat.In group C and D,in the same period of 5 and 7 days after operation,compared with group B,the difference was significant(P<0.05).On the 1st day after operation,There was a significant statistical differences between the three groups of B,C and D(P>0.05).3.Detection of peripheral splenic lymphocytes by flow cytometry:Id and 3d after operation,there was no difference on the expression of CXCR4 in spleen lymphocytes between groups B,C,D(P>0.05),and there was significant difference with group A(P<0.05).At 5d and 7d after operation,groups C and D were significantly lower than group B,and the difference was significant(P<0.05).4.Morphological examination of transplanted liver:The degree of transplanted liver AR was graded by using Banff classification,In group A,the tissue structure of the transplanted liver was normal at each time point after operation.On the 1st day,the hepatic lobule structure is basically intact in groups B,C,D.The hepatocytes were basically free of degeneration and necrosis.and there was only a little inflammatory cells infiltration in the portal area,and no rejection occurs.On the 1st day after transplantation,the rejection was unclear in group B,and mild(Class Ⅰ),moderate(Class Ⅱ),and severe(Class Ⅲ)rejection occurred on days 3,5,and 7 after surgery.Mild AR occurred in a small number of tree shrews within 3 days to 10 days in group C and D after surgery.The rejection of group B was significantly more serious than group C and D in the same period(P<0.05).Acute rejection could be clearly diagnosed on the 3rd day in group B after operation.5.Immunohistochemical detection of liver CXCL12 expression:On the 7th day after surgery,CXCL12was most expressed in liver cells,and the expression of CXCL12 was less in group C and group D,compared with group B there was no difference(P<0.05).In group A,the normal control group CXCL12 was also expressed,but the amount was small.Group B,C,D compared with group A,there was a significant difference(P<0.05).6.Detection of CXCL12 protein expression in peripheral blood of tree shrew by enzyme-linked immunosorbent assay:There was no difference on serum CXCL12 between group B,C and D on the 1st day after liver transplantation(P>0.05),but compared group A with group B,C,D,there was significantly difference(P<0.01).After 3d transplantation,serum CXCL12 in group B increased rapidly,which was significantly different from the expression level of Id before surgery(P<0.05),and was upregulated with time.The expression levels of group C and D increased slowly on the 5th day and 7th day after operation,and the expression level was lower when compared with group B(P<0.05).The expression change of CXCL12 protein was positively correlated with RAI,and the correlation coefficient(r)was 0.880.7.Western blot analysis:There was no Statistical difference on the expression of CXCR4 protein in the transplanted livers in each group on the 1st day after transplantation.The expressions of CXCR4 protein in groups A,C and D was significantly lower than that of group B on the 3rd,5th and 7th day after operation(P<0.05).The expressions of CXCR4 protein in group C and D was higher than that in group A(P<0.05),but there was no significant difference in CXCR4 protein expression between group C and D on the 3rd,5th and 7th day(P>0.05).[Conclusion]1.Tree shrew has a good homology with human beings,and human-related reagents can be applied to the experimental study of tree shrew.The reliability of the results of this experiment further proves that the liver transplantation model of tree shrew is a kind of experimental study that can be applied to the whole process of liver transplantation,which has unique advantages and values.2.Using a model of liver transplantation in tree shrew show that CXCL12 expression was found in normal liver tissues.In acute rejection,lymphocytes and inflammatory cells invasion can occur in combination with CXCR4,causing the transplanted]ive dysfunction or damage.3.Chemokine receptor inhibitor AMD3100 can block the mechanism of CXCL12/CXCR4 axis in liver transplantation rejection and alleviate acute rejection.The mechanism may be to reduce the infiltration of T lymphocytes into the graft and inhibiting the activation.4.Rapamycin inhibited lymphocyte migration induced by human recombinant protein CXCL12 in tree shrews.The possible mechanism is that down-regulation of CXCL12,affects the expression of CXCR4 receptor,thereby affecting lymphocyte migration mediated by CXCL12/CXCR4 signaling pathway and exerting immunosuppression.5.The expression level of CXCL12/CXCR4 after liver transplantation was closely related to the degree of acute rejection,and the expression level was related to the intensity of rejection,which could be used to evaluate the state of immune rejection.6.The sustained high expression of CXCL12 in serum can be a sensitive predictor of acute rejection of liver transplantation.Detection of serum CXCL12 expression can be used as a noninvasive and sensitive method for early diagnosis of acute rejection of liver transplantation,which provides reference for clinical monitoring and diagnosis.The level of CXCL12 expression can predict the severity of rejection and evaluate the therapeutic effect of anti-rejection. |
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