The Role Of Transcription Factor YY1 In Acute Rejection Of Liver Transplantation In Rats

Research background and objective:Liver transplantation is the first choice for the treatment of end-stage benign liver disease.Immune rejection is the bottleneck restricting the survival of donor liver after liver transplantation.Helper T 17 cells(Th17)and regulatory T cells(Treg)play a key role in immune rejection.The former promotes immune rejection,while the latter maintains immune tolerance.Yin-Yang 1(YY1),as a nuclear transcription factor,plays a role in many biological processes.It was found that YY1 could promote the differentiation of Th17 by activating the transcriptional expression of interleukin-6(IL-6)and blocking the Forkhead box protein p3(Foxp3)to inhibit the differentiation and function of Treg,resulting in Th17/Treg cell imbalance.However,the effect of YY1-mediated Th17/Treg cell imbalance on the immune rejection of liver transplantation remains unknown.Therefore,this study aims to observe the expression changes of YY1 in the donor liver of the acute rejection model of orthotopic liver transplantation in rats,overexpress the plasmid of YY1,induce and culture the dendritic cells(DCs)derived from bone marrow of Lewis rats,and classify the CD4~+T lymphocytes from spleen of BN rats by magnetic beadseparation.Furthermore,the role of YY1 in immune rejection of liver transplantation is discussed to provide relevant ideas and theoretical basis for clinical diagnosis and treatment.Method:1)Establishment of rat orthotopic liver transplantation model:modified Kamada’s"two-sleeve method"was adopted.Sham group:Brown Norway rats(BN rats)without liver transplantation were used.Syngraft group:The donors were BN rats and the recipients were BN rats.Allograft group:Lewis rats were the donors and BN rats were the recipients.Donor liver tissues and serum were collected 5 days and 10 days after operation.Morphological observation was performed on the donor liver tissues.Immunohistochemical staining(IHC)was used to detect the expression of CD3.The levels of IL-6 and Tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA).2)Proteomic analysis was performed on donor liver tissue from 5 allogeneic and 3isogeneic groups at 10 days postoperatively.The quality control of the samples was carried out using quantitative protein running,pre-mass spectrometry database comparison and labeling efficiency as the quality control points.The differentially expressed proteins were screened when the protein expression difference ratio between groups was greater than 1.2 and P<0.05.3)According to the results of protein expression differences between the screened groups,the transcription factor YY1 was selected as the research object of this experiment.IHC and Western blot(WB)were used to detect the expression of YY1 in the transplanted liver tissue of the sham operation group,the syngeneic transplantation group and the allogeneic transplantation group at 5 days and 10 days after surgery.Quantitative polymerase chain reaction(qPCR)was used to detect the mRNA expression level,and IHC was used to detect the expression of CD86.4)YY1 overexpression plasmid was constructed,and 293T cells were used to package lentivirus,and then virus concentration and titer determination were carried out.5)DCs from bone marrow of Lewis rats and CD4~+T lymphocytes from spleen of BN rats were cultured in vitro.Im DCs were infected with YY1 overexpressed lentivirus by inverted microscope,and then phenotypic identification was performed by flow cytometry.The contents of cytokines in Th1 and Th17 cells were detected by flow cytometry.Results:1)The rat orthotopic liver transplantation model was successfully constructed.On5 days and 10 days after surgery,the allogeneic transplantation group showed acute rejection changes,CD3~+T lymphocytes in the vascular region were obviously infiltrated,and the expressions of AST,ALT,IL-6 and TNF-αin serum were higher than those in the syngeneic transplantation group(P<0.05).2)The results of proteomic analysis showed that the protein quality was good,the total amount was sufficient,and the parallelism between samples was good.The enzymatic hydrolysis and the behavior of chromatography-mass spectrometry were normal,which could be used for subsequent difference analysis.A total of 3,276differentially expressed proteins were screened according to the criteria of protein expression difference ratio greater than 1.2 and P<0.05.Compared with the syngeneic transplantation group,there were 2,175 differentially expressed proteins up-regulated and 1,101 differentially expressed proteins down-regulated in the allogeneic transplantation group.YY1 protein was upregulated in the allogeneic transplantation group,with a differential expression ratio of 1.255 and P=0.00307.3)At 10 days after orthotopic liver transplantation,the mRNA expression level of YY1 in the allogeneic transplantation group was higher than that in the syngeneic transplantation group and the sham operation group(P<0.05),and the protein expression level of YY1 at 10 days after operation in the allogeneic transplantation group was higher than that in the syngeneic transplantation group and the sham operation group(P<0.05).The positive IHC expressions of YY1 and CD86 in allogeneic transplantation group were higher than those in syngeneic transplantation group and sham operation group at 5 days and 10 days after operation.4)The constructed YY1 overexpressed plasmid was sequenced and the gene sequence was completely correct.After lentivirus packaging,titer determination was performed,and the results were 5×10~8TU/m L and 1×10~9TU/m L.5)DCs from bone marrow of Lewis rats were isolated and cultured.The infection efficiency of lentivirus infection with YY1 overexpression was about 80%,and the optimal MOI value was 15.Flow cytometry showed:The expression of CD80,CD86and MHCⅡon the surface of m DCs were higher than those on im DCs.The expression of CD80,CD86 and MHCⅡin the overexpressed plasmid of YY1 was higher than that in the empty vector group and PBS group.Flow cytometry was used to detect the intracellular cytokines associated with Th1 and Th17 cells,and it was found that DCs infected with YY1-overexpressing lentivirus could promote the differentiation of CD4~+T cells from the spleen of BN rats toward Th17.Compared with empty vector group and PBS group,the secretion of IL-17 and IFN-γin YY1-overexpressing plasmid group was increased(P<0.05).Conclusions:1)Lewis rats as donors and BN rats as recipients can successfully construct the rat orthotopic liver transplantation model.2)The expression level of YY1 in donor liver of rat liver transplantation was significantly increased.3)YY1 may induce acute rejection by promoting the differentiation of Th17 cells.