Experimental Study On HBV Specific CTL Reaction Induced By HBcAg Mediated By TPP?

Aim:To investigate the effect and mechanism of HBcAg on HBV specific CTL response induced by TPP?,The effect of HBcAg on the signal molecules of JAK/STAT signal pathway under TPP?.To determine the key step its activation of CTL response.To provide a new strategy and experimental basis for clinical treatment of chronic hepatitis B antiviral therapy.Materials and methods:1.HBcAg and TPP? gene fragments were amplified by PCR,Gene fragment was inserted into adenovirus vector to construct recombinant adenovirus expression vector,Liposome was used to transfect the recombinant adenovirus expression plasmid,plasmid and envelope plasmid into 293T cells.The recombinant adenovirus particles containing HBcAg and TPP? gene Adv-HBcAg and Adv-HBcAg-TPP? were obtained for further purification and identification.2.DC cells were isolated and cultured in vitro and transfected into DC cells with different recombinant adenovirus.Detection of IL-12 secretion in supernatant of DC cells by ELISA.Flow cytometry was used to detect the expression of CD80,CD83,CD86 and MHC I molecules on the surface of DCs.Detection of IL-2,L-4,IL-10 and IFN-in the supernatant of T cells by ELISA.Flow cytometry was used to detect the number of CD8~+T+IFN-?~+T cells.T lymphocyte proliferation was detected by CCK-8assay and lactate dehydrogenase release assay was used to detect the specific CTL reaction.3.Mice were randomly divided into 6 groups,respectively:empty virus group(control group);HBcAg recombinant adenovirus group;Adv-HBcAg and blocker group(AG490);Adv-HBcAg-TPP? recombinant adenovirus group;Adv-HBcAg-TPP? recombinant adenovirus and blocker group(AG490)group.Detection of T lymphocyte factor secreting cytokines by ELISA.Flow cytometry and enzyme-linked immunosorbent assay for the detection of T lymphocytes.Detection of the expression of JAK/STAT signaling pathway by Real-time PCR and Western blot.4.HBV transgenic mice were randomly divided into 6 groups:the experimental group Adv-HBcAg-TPP? immune group;the control group was Adeno immune group,Adv-HBcAg immune group,HBcAg group;20000IU IFN-group and PBS group.Mice were immunized four times.The liver tissues,serum and spleen tissues were collected.The release of T lymphocyte was detected,the activity of CTL was detected,the proliferation of T lymphocytes was detected,and the levels of T lymphocytes were detected.Results:1.HBcAg and TPP? gene were successfully inserted into the adenovirus vector and the target protein was successfully expressed in 293T cells detected by Western blot method.2.Recombinant adenovirus Adv-HBcAg-TPP? can promote the expression of DC molecules on the surface of CD80,CD83,CD86 and MHC,and can also stimulate the release of IL-12 from DC cells.Adv-HBcAg-TPP? can promote the secretion of IL-2and IFN-?,and recombinant adenovirus Adv-HBcAg-TPP? can enhance the expression of CTL.3.Compared with the other groups,the levels of IFN-?(P=0.031)and IL-2(P=0.015)increased significantly.The number of T lymphocytes in the CD8?~+IFN-?~+and CD4~+IFN-?~+was significantly higher than in other groups(Pvalue was 0.00 and 0.012respectively).The lymphocyte proliferation activity was significantly higher than that of other groups(P=0.00),and there was no significant difference before and after the antagonist AG490(P=0.521).The Adv-HBcAg-TPP? group was significantly higher than that of Adv-HBcAg group(P=0.041).The expression of mRNA of JAK2,Tyk2,STAT1 and STAT4 mRNA in Adv-HBcAg-TPP? group was higher than that in other groups,and the changes of these molecules in protein level also showed a similar trend with mrna level.4.Compared with Adv-HBcAg group,Adeno group,HBcAg group,IFN-?group and PBS group,The inhibition rate of HBV DNA in Adv-HBcAg-TPP? group was significantly higher(P=0.021),The level of serum AST and ALTwas higher than that of other groups(P=0.01).Normal morphology and lobular structure of hepatocytes in PBS group and adeno group,portal area and central jingmaiqu inflammatory lymphocytes quantity small,The number of portal area and inflammatory cells in the liver of the HBcAg group and the central group increased,and thecell infiltration was the most obvious in adv group.Liver tissue of PBS and Adeno micethe expression of HBcAg and HBsAg showed the characteristics of diffuse distribution,and the expression of diffuse,After the immunization of HBcAg andAdv-HBcAg-TPP?,the expression of HBcAgand HBsAg decreased gradually.Potential,the decrease of HBsAg and HBsAg in the group group was the most significant;IFN-?and IL-2 level was higher than that in other groups.CD8?~+IFN-?~+and CD4~+IFN-?~+was significantly higher than in other groups.The proliferation activity of lymphocytes in adv group was higher than that in HBcAg group,Western blot analysis expression of T-bet,Adv-HBcAg-TPP? group was significantly higher than that in HBcAg group and PBS group,GATA group,the ratio of bet/GATA increased significantly.Changes of T-bet/GATA-3 may be involved in the immune response of the above.Conclusion:1.The recombinant,packaged and purified adenovirus Adv-HBcAg-TPP? was successfully constructed.At the same time,the recombinant adenovirus Adv-HBcAg was successfully constructed and packaged.The protein was stably expressed in transfected 293T cells.2.Recombinant adenovirus Adv-HBcAg-TPP? can promote the maturation of DCs cells,enhance the proliferation of T lymphocytes and induce strong HBV specific CLT immune response.3.Recombinant adenovirus Adv-HBcAg-TPP? immune HBV transgenic mice,stimulate the secretion of factor T lymphocyte T H1 like cells,promote the proliferation and activity of specific CTL T lymphocytes and activation of JAK/STAT signaling pathway may be involved in the immune response of the above changes.4.Recombinant adenovirus Adv-HBcAg-TPP? immune HBV transgenic mice,stimulate the secretion of factor T lymphocyte T H1 like cells,promote the proliferation and activity of specific CTL T lymphocytes,changes of T-bet/GATA-3may be involved in the immune response of the above.