Mechanism Research Of ZAP70 On HSP65 Mediated Proliferation And Reverse Cholesterol Transport In T Lymphocytes

BackgroundAtherosclerosis is the underlying cause of the cardiovascular event,and its pathological basis including abnormal lipid metabolism,vascular endothelial cell injury and vascular chronic inflammation.A variety of cells derived from innate and adaptive immune system can promote the development of atherosclerosis.T cells in atheromatous plaque can recognize autoantigen caused a series of immune inflammation,release a variety of inflammatory factor aggravating inflammation promote the atherosclerosis formation.Heat shock protein 65 is a highly conserved protein,has strong immunogenicity,vulnerable to identify the body's immune system.It has been confirmed that heat shock protein 65 can interact with T cell receptor to induce strong immune inflammatory response.Activated T lymphocytes can promote the occurrence and development of AS by releasing various metabolic enzymes,toxins and inflammatory signaling pathway proteins.ZAP70 is the key regulating points of TCR signaling pathways,can affect the physiological function of T lymphocytes.Our previous study showed that subcutaneous immunization with heat shock protein 65 can promote the inflammatory response,which can damage HDL function and accelerate the development of atherosclerosis.However,whether HSP65 affects the proliferation of T lymphocytes,the activation of immune inflammatory pathway and the reverse cholesterol transport function through ZAP70 signaling pathway has not been reported at home and abroad.ObjectivesZAP70 positive Jurkat T lymphocytes and ZAP70 negative P116 T lymphocytes as the research object,study the role of ZAP70,the key regulator of the TCR signaling pathway,in HSP65 mediated T lymphocyte proliferation and reverse cholesterol transport function.At the same time,we used lentivirus shRNA-ZAP70 to transfect Jurkat T lymphocytes to block the ZAP70 signaling pathway,and further verify the HSP65 affect the mechanism of action of T lymphocytes physiological activities.First PartMethods1.CD4+T lymphocytes and Jurkat T lymphocytes were stimulated by HSP65,CCK-8,WST-1 and cell cycle assay were used to detect the proliferation of T lymphocytes.2.CD4+T lymphocytes and Jurkat T lymphocytes were stimulated by HSP65,detection of cholesterol efflux rate by[3H]-cholesterol.Results1.HSP65 stimulated T lymphocytes can promote the proliferation of T lymphocytes.2.HSP65 stimulated T lymphocytes can reduce cholesterol efflux rate.Second PartMethods1.Lentiviral transfection of Jurkat T lymphocytes blocked the ZAP70 signaling pathway,and the lentivirus transfection effect was detected by flow cytometry,and the silencing effect of lentivirus was detected by WB and qPCR.ZAP70 protein expression level of P116 T lymphocyte was detected by WB techniques.2.Jurkat T lymphocytes were stimulated by HSP65,and the expression of ZAP70 protein phosphorylation was detected by WB.3.Blocking ZAP70 signaling pathway,WST-1 assay were used to detect the proliferation of T lymphocytes under HSP65 stimulation.4.Blocking the ZAP70 signaling pathway,using[3H]-cholesterol to detect the effect of HSP65 mediated T lymphocyte cholesterol efflux rate,and the use of WB and qPCR detection of cholesterol transport related proteins and mRNA.5.Blocking ZAP70 signaling pathway,using WB and immunofluorescence detection of nuclear factor NF-kappa B activation of T lymphocyte stimulated by HSP65.Results1.HSP65 stimulates T lymphocytes increase ZAP70 phosphorylation.2.Blocking ZAP70 reduces HSP65 mediated and non HSP65 mediated Jurkat T lymphocyte proliferation.3.Blocking ZAP70 increased HSP65 mediated and non HSP65 mediated Jurkat T lymphocyte cholesterol efflux rate and level of cholesterol transport related proteins.4.Blocking ZAP70 reduces HSP65 mediated and non HSP65 mediated NF-kappa B activation of Jurkat T lymphocyte.Conclusions1.HSP65 can activate ZAP70 of T lymphocytes.2.Blocking ZAP70 inhibit the proliferation of HSP65 mediated T lymphocytes.3.Blocking ZAP70 can increase HSP65 mediated cholesterol efflux rate and expression of transporter protein.4.Blocking ZAP70 inhibits HSP65 mediated NF-kappa B activation in T lymphocytes.5.Through the study,we have shown that blocking ZAP70 can increase HSP65 mediated reverse cholesterol transport function of T lymphocytes,inhibit cell proliferation and activation of NF-kappa B.