The Influence Of The Express Of TLR9 And IRF7 Imposed By HBcAg Recombinant Adenovirus's Transfection Of Human Peripheral Blood Mononuclear Cells

Objective:Adenovirus is used as a carrier to load HBcAg gene into peripheral blood mononuclear cells (PBMCs) of healthy donors and chornic HBV carriers. The expression of TLR9 and IRF7 as well as the secretion of IFN-a were detected before and after the transfection so as to learn the changes of immune recognition state of chronic HBV infector.Method:Carrying HBcAg gene is constructed to replication defective recombinant adenoviral. And it is used to transfect PBMCs of the healthy donors and chronic HBV carriers. Then they were stimulated with high copy HBV-DNA serum and/or CpG-ODN2006 by group,RT-PCR method was used to detect mRNA expression of TLR9 and IRF7 before and after the ransfection,Western-Blot method was used to detect protein level expression of TLR9 and IRF7, and Elisa method was used to detect the level of IFN-a of culture medium.Result:â‘ The RT-PCR result showed that TLR9 and IRF7 mRNA expression of the chronic HBV carriers before transfection is lower than healthy donors (P<0.05). The TLR9 and IRF7 mRNA expression of chronic HBV carriers and healthy donors did not show a significant increase when they were stimulated by CpG-ODN2006 before transfection(P>0.05). However, The TLR9 and IRF7 mRNA expression of chronic HBV carriers and healthy donors did show a significant increase when they were stimulated by CpG-ODN2006 after transfection, there is statistics difference (P<0.05)â‘¡Consistent with the mRNA expression of TLR9 and IRF7, the Western-Blot result also showed that the protein expression of TLR9 and IRF7 of healthy donors was higher when they were stimulated after transfection, there is statistics difference (P<0.05). In comparison with the status before transfection, the protein expression of TLR9 and IRF7 of chronic HBV carriers was higher after transfection and there is statistics difference (P<0.05) â‘¢The Elisa result showed that the secretion of IFN-a of chronic HBV carriers was lower than healthy donors before and after transfection (P<0.05, P<0.05). The secretion of supernatant of IFN-a of chronic HBV carriers and healthy donors was increased significantly before transfection and there is statistics difference (P<0.05)Conclusion:â‘ Compared with the status before transfection, the mRNA of TLR9 and IRF7 and the protein expression as well as the secretion of IFN-a in PBMCs of chronic HBV carriers were lower than those of healthy donors, which suggests that the ability to recognize HBV nucleic acids DNA by the PBMCs of chronic HBV carriers was lower, IRF7 could not be activated effectively and IFN-a was not produced sufficiently to eliminate HBV, which may cause chronic persistent infection.â‘¡After Ad-HBcAg was transfected by PBMCs of chronic HBV carriers, the secretion of IFN-a was lower than healthy control group, which suggests that the HBV persistent infection is connected with the decrease of immune function of PBMCs.â‘¢After Ad-HBcAg was transfected by PBMCs, the mRNA and protein expressions of TLR9 and IRF7 as well as secretion of IFN-a were higher than before transfection, which proved that TLR9 is an intracellular receptor, which can produce strong anti-HBV immune with intracellular stimulation.â‘£After the transfection, CpG-ODN can activate the expression of TLR9 of IRF7 in PBMCs of chronic HBV carriers. Meanwhile, the secretion of IFN-a was enhanced, which suggests that CpG-ODN can be used to fight against HBV through innate immune system.