Establishment Of Regression Model Of Differential Gene Expression In Liver Transplantation And Skin Transplantation Rejection In Rats

Background:Liver transplantation is an effective treatment for end-stage liver disease.At present,the diagnosis of rejection after liver transplantation is mainly carried out by needle biopsy,but some patients have poor coagulation function after transplantation,which is at great risk.Whether a simple and effective method can be used to detect the degree of liver rejection and make corresponding management is crucial to the long-term prognosis of patients after transplantation.Currently in liver transplantation rejection can be achieved by intrahepatic rejection degree of gene diagnosis,because the transplanted skin in the skin graft rejection reaction can be observed through the surface,mostly in the symptoms after symptomatic treatment,found in joint transplantation through in the small intestine transplantation transplantation skin at the same time,can use the skin transplant prediction of small intestine rejection,However,the correlation between liver rejection and other organs has not been reported,and the correlation regression between skin transplantation and liver transplantation rejection remains to be solved.The first part: Rapid establishment of rat model of liver transplantation by portal vein priority reverse anastomosisResearch Background:The traditional "two-cuff method" rat liver transplantation model takes a long time to establish and is difficult to train,so it is difficult to adapt to the current training mode of postgraduates.Objective:The model of rat liver transplantation can be established quickly by using the characteristics of multiple steps in establishing rat liver transplantation model,reasonably combining the time and method of each step,shortening the learning time and reducing the difficulty of operation.Methods:Based on the "two-sleeve method",the portal vein was prefered for cannula anastomosis on the basis of maintaining smooth venous blood flow in the inferior cavity.The cannula was reversed anastomosis with donor liver placed in the lower abdomen of rats(portal vein priority group),and the rat liver transplantation model was established and compared with the traditional rat liver transplantation model(traditional transplantation group).Results:The learning time of portal vein priority group was significantly better than that of traditional transplantation group.Conclusion:The establishment of rat model of portal vein priority liver transplantation can shorten the learning period,reduce the difficulty of operation,and provide a reliable method for beginners to quickly master the establishment of rat liver transplantation model,which is suitable for learning and promotion.The second part: Rna-seq analysis and VALIDATION of LCK,LAT and ZAP-70 in rat liver transplantation rejectionBackground:The prevention and treatment of rejection after liver transplantation is still a key clinical issue.Objective:To investigate the pathophysiological mechanism of liver transplantation rejection in rats,and to select candidate genes to provide clues for identifying the degree of rejection and potential therapeutic targets.Methods:The Brown Norway(BN)-BN transplant tolerance model(N =3)and lewi-BN transplant rejection model(n=3)were established.Specimens were obtained 7 days after transplantation,venous blood was collected for liver function testing,liver tissue was subjected to HE staining and RNA-seq.Differential gene screening after sequencing,KEGG and GO enrichment analysis,real-time quantitative PCR detection,to determine the accuracy of RNA-SEQ results.Lymphocyte-specific protein Tyrosine kinase(LCK),T cells activate Connexins(LAT),ζ Verification of Ze TA-chain(TCR)associated protein kinase 70kDa(ZAP-70)protein.Results:1.Compared with the tolerance group,the liver function of the rejection group was poor.According to Banff score,the rejection group showed severe rejection,indicating that the liver transplantation tolerance and rejection model had been established successfully.2.A total of 19186 genes were identified in rat liver tissue samples,and 7521 Differential genes(DEGs)were found by comparing the genes of the exclusion group and the tolerance group(P < 0.05).In which the changes of 6413 genes(| log2(Fold Change)| > 1,p < 0.05),the rejection has raised 3355 gene expression,gene expression by 3058.KEGG results showed that 8 pathways were related to immune system process,among which 7 up-regulated genes were enriched,and 1 down-regulated genes were enriched.Further screening of 289 genes was carried out,and 147 genes were screened by IMMPORT database.Found 97 immune genes | log2 Fold Change | > 2,GO enrichment analysis results show that raising genes involved in the process of immune response,lower gene mainly play a role in the metabolic process,on the biological functions involved in the body’s defense reaction.3.According to rn A-SEQ analysis,the expressions of some genes were verified by Realtime PCR(RT-PCR).The study found that the two genes had similar trends,and the protein expressions of LCK,LAT and ZAP-70 were consistent with the RNA-SEQ results.These proteins were positively expressed on inflammatory cells in the sinusoids and sinks of the liver in the rejection group.Conclusion:1.CD86,C3,PROC,C5,LCK,LAT,ZAP-70 and other immune rejection related genes were found to lead to the occurrence of liver transplantation rejection.2.The functions of LCK,LAT and ZAP-70 proteins may affect the progression of liver transplantation rejection,and can be used as candidate markers for detecting the degree of liver transplantation rejection.The part three:Screening and analysis of RNA-SEQ differential gene expression in skin graft rejectionResearch Background:There is no accurate method to predict the degree of rejection after skin transplantation.How to accurately predict and diagnose the degree and development of rejection is an urgent problem to be solved.Objective:Through the establishment of rat skin transplantation model,high-throughput gene sequencing was carried out,and sequencing technology was applied to analyze and search for genes and diagnostic markers related to rejection reaction.Methods:Lewi-lewis skin graft tolerance model(n=4)and Lewi-Bn skin graft rejection model(n=3)were established.The grafted skin tissue was collected 7 days after transplantation for RNA-SEQ sequencing to analyze the expression of differential genes.KEGG and GO enrichment analysis of differential genes was performed to select candidate genes for diagnosis of rejection.Results:1,in the rat skin grafts tissue samples,a total of 20997 genes were identified,the rejection and tolerance group DEG contrast,found that there are 5109 different genes(p 0.05)or less,of which 1088 gene changes(| log2(Fold Change)| > 1,p 0.05)or less,In the rejection group,734 genes were up-regulated and 354 genes were down-regulated.2.KEGG pathway enrichment analysis showed that up-regulated gene pathways were enriched,with 9 pathways enriched in immune system process and down-regulated gene pathways enriched,most of which were enriched in signal transduction and molecular regulation.3.GO enrichment results confirmed that cell adhesion was related to the process of rejection.The possible diagnostic markers of rejection were Granzyme B(GZMB),Chemokine CXCL9 and Cytotoxic T lymphocyte-associated protein 4(CTLA4).Conclusion:Rna-seq sequencing of skin transplantation revealed that three immune genes,GZMB,CXCL9 and CTLA4,may play an important role in skin rejection,which provides a basis for further exploration of immune rejection after transplantation.The fourth part : Regression model of differential gene expression related to liver transplantation and skin transplantation rejectionResearch Background:Simple and accurate detection is very important for the rejection after liver transplantation.However,the correlation between skin graft rejection and liver transplantation rejection and their correlation remains unclear.Objective:To investigate whether the gene expression level of liver graft rejection can be determined by skin graft gene expression so as to guide the diagnosis and treatment of liver graft rejection.Methods:According to the second and third parts of liver transplantation and skin transplantation rn A-SEQ differentially expressed genes,the homonymous genes were compared,and the differentially expressed genes with the same expression were screened out for curve estimation,so as to obtain the optimal model and establish the regression model.Results:A comparative analysis of the same name showed that 806 genes were different in both Liver transplant acute rejection(LTAR)and Skin graft acute rejection(STAR).It was found that the expression levels of differential genes in the rejection group were correlated,with the correlation coefficient R =0.795,R2=0.632.The regression equation was as follows: the expression level of LTAR gene in liver = the expression level of STAR gene in skin ×6.914+233.484.Among them,155 homologous immune genes were further screened,and correlation analysis was performed,with r=0.940,R2=0.883.The regression equation was: LTAR gene expression level in liver = STAR gene expression level in skin ×6.119+431.467.At the same time,16 genes were highly expressed in T cells or macrophages,with correlation coefficient R =0.865,R2=0.748.The regression equation was as follows: liver LTAR gene expression level = skin STAR gene expression level ×3.048+840.874.Conclusion:The gene expression level of transplanted liver can be accurately predicted by the gene expression level of transplanted skin,which has important guiding significance for the diagnosis and treatment of liver transplantation rejection.Summary:1.Portal vein priority reverse anastomosis technique provides a relatively fast and reliable method for beginners to quickly master the establishment of rat liver transplantation model,which is suitable for learning and promotion.2.LCK,LAT and ZAP-70 are up-regulated in liver transplantation rejection and may play an important role in the rejection.3.In skin transplantation,GZMB,CXCL9,CTLA4 and other genes may participate in the immune response process of rejection and play an important role in it.4.The correlation regression model between skin rejection gene expression level and liver transplantation rejection gene expression level was established to supplement the existing deficiency of liver transplantation rejection detection and provide theoretical basis for the diagnosis and treatment of liver transplantation rejection.