A Study On Effect Of TIPE2 Gene Modified Human Amniotic Mesenchymal Stem Cells To Ischemia-reperfusion Inflammatory Injury In Homologous Mouse Heart Transplantation

Objective:The aim of the study was to investigate the effect of tumor necrosis factor-αinduced protein 8-2(TIPE2)gene modified human amniotic mesenchymal stem cells(h AMSCs)on ischemia-reperfusion inflammatory injury in homologous mouse heart transplantation and the potential mechanism.Moreover,it was proved that TIPE2 gene modified h AMSCs had better anti-inflammatory effect than h AMSCs.It provides research experience for the further study and application of TIPE2 gene modified h AMSCs in the field of heart transplantation,and also brings new ideas for the application of h AMSCs gene modification research in the field of heart transplantation.Methods:1.Vivo experiments:The model of myocardial ischemia-reperfusion injury after neck heart transplantation in homologous mice was established.Experimental groups were divided into control group(single heart transplantation group),h AMSCs group and TIPE2-h AMSCs group.After modeling,the same volume of phosphate buffer salt solution(PBS)solution,5×10~5h AMSCs/0.2m L and 5×10~5TIPE2-h AMSCs/0.2m L were injected into each group through the tail vein,respectively.Samples were collected 24 hours after injection,and hematoxylin-eosin staining(HE)was used to observe the myocardial pathological changes in the right ventricle of donor heart.The levels of interleukin-8,interleukin-10 and tumor necrosis factor-αin peripheral blood were determined by ELISA.The levels of IL-8,IL-10 and TNF-αin myocardial tissue were determined by immunohistochemistry,and the expression of p38 mitogen-activated protein kinase in myocardial cells was detected by western blotting.2.Vitro experiments:The model of myocardial ischemia-reperfusion injury after neck heart transplantation in mice was established.Peripheral blood of the recipient was collected,and peripheral blood mononuclear cells(PBMC)were isolated and extracted by Ficoll density gradient centrifugation.The experimental groups were divided into control group(PHA+PBMC),h AMSCs+PHA+PBMC co-culture group and TIPE2-h AMSCs+PHA+PBMC co-culture group.The proliferation of PBMC was observed 24 hours later,and levels of IL-10 and TNF-αwere detected by ELISA.Results:1.Vivo experiments:(1)Myocardial pathology of the right ventricle:myocardial cells in the control group showed edema,lymphocytes and red blood cells infiltrated in muscle space,and monocytes were seen locally.In the h AMSCs group,some myocardial cells showed mild edema,a small number of lymphocytes and red blood cells infiltrated in the muscle space,and monocytes and neutrophils were seen locally.In the TIPE2-h AMSCs group,some myocardial cells in the right ventricle showed mild edema,and scattered lymphocytes and red blood cells were observed in the muscle space.(2)IHC of myocardial tissue and peripheral blood ELISA:Compared with the control group,levels of IL-8 and TNF-αin h AMSCs group and TIPE2-h AMSCs group were down-regulated and levels of IL-10 were promoted,and the effect of TIPE2-h AMSCs group was better than h AMSCs group.(3)WB detection of p38 protein in myocardial tissue:p38 protein expression decreased in both h AMSCs group and TIPE2-h AMSCs group.2.In vitro experiments:(1)Flow cytometry test:PBMC showed significant proliferation after PHA stimulation.Compared with the control group,both h AMSCs and TIPE2-h AMSCs inhibited PBMC cell proliferation,and TIPE2-h AMSCs inhibited PBMC cell proliferation better than h AMSCs.(2)ELISA showed that both h AMSCs and TIPE2-h AMSCs down-regulated TNF-αlevel and promoted IL-10 level,and the effect of TIPE2-h AMSCs was better than h AMSCs.Conclusion:1.Combined with the results of myocardial histopathology,peripheral blood ELISA,immunohistochemistry,flow cytometry and ELISA in vitro experiments.It was shown that h AMSCs could alleviate the inflammatory injury caused by ischemia reperfusion in homologous mice heart transplantation,and TIPE2-h AMSCs had a better inhibitory effect than h AMSCs.2.The inhibitory effect of h AMSCs and TIPE2 gene modified h AMSCs on ischemia-reperfusion injury of homologous heart transplantation may be potentially associated with the expression of p38 protein.