| BackgroundViral hepatitis type B caused by hepatitis B virus (HBV) is one of infectious diseases that are serious harm to human health. HBsAg carriers in China have been more than 120 million, and 30 million people are chronic hepatitis B carriers, about 1-1.5 million people each year die from acute or chronic HBv infection. Up to now, there is still no effective treatment for such patients, leading to persistent disease in patients, and some patients eventually develop to cirrhosis or liver cancer, which brings serious social and family burden. At present, a comprehensive treatment mainly through the anti-virus, immune regulation, improving liver function and anti-liver fibrosis is used for the treatment of chronic hepatitis B, but the effect is not satisfactory. Therefore, hepatitis B vaccination is an important measure to prevent and control the spread of hepatitis B virus. However, there are still some problems for the Hepatitis B vaccine: firstly, the existing vaccines can not break the body's immune tolerance of hepatitis B or hepatitis B virus carriers to make it clear virus; secondly, HBsAg variants that are likely to avoid the protective effect of current vaccines are still an issue; thirdly, about 5% ~ 10% of hepatitis B immunization people do not produce protective antibodies, or antibody level is very low.Hepatitis B DNA vaccine developed in recent years is expected to solve these problems. As the DNA vaccines have both the advantages similar to live attenuated vaccine, and the safety of inactivated vaccine or subunit vaccine, they have the dual roles of disease prevention and therapeutic effect. Although experienced rapid and extensive development in recent years, the overall immune effect of the DNA vaccines is still not satisfactory. For example, some DNA vaccines work well in the small animal immunization experiments, but they are not so effective in large animal experiments; some DNA vaccines can play a very good immune protection in animal experiments, but they can not protect subjects against pathogens attack in clinical trials. Therefore, it is a Confronting scientific challenge to enhance the immune effect of DNA vaccines. Developing new type of adjuvant and controlled release formulation that they can stimulate cell-mediated immunity to form a stronger therapeutic treatment of hepatitis B virus vaccine are two promise ways. Naringenin has the effect of antibiosis, anti-inflammatory, anti-cancer, spasmolysis, and choleretic effect. A protective effect of naringenin on acute immunological liver injury has been reported. However, it is seldom reported whether naringenin is able to enhance the immune effect of hepatitis B DNA vaccine. The study has been focused on whether naringenin can be used as a new type of hepatitis B DNA vaccine adjuvant, and the immune mechanism that naringenin enhance the immune effect of hepatitis B nucleic acid vaccine has been discussed in detail.In addition, previous studies have confirmed that PEGylation of peptide or protein can delay its half-life, increase its water solubility and stability. However, there is yet no research on whether it has a delayed effect on the plasmid DNA. In this thesis, polyglycol has been studied as a delivery system for the immune effect HBV DNA vaccine.ObjectIn this study, the impact of naringenin as an adjuvant on the hepatitis B DNA vaccine was evaluated by detecting the antibody level, the proliferation effect of T cell and the response of cytotoxic T lymphocyte. The mechanism of naringenin enhancing effect of hepatitis B DNA vaccine was researched by detection of the cytokines. At the same time, it was also studied whether PEG as a carrier transmission system can enhance the humoral and cellular immunity of HBV DNA vaccine. MethodThe eukaryotic expression vector pVAX-S2 containing HBV pre-S2 and S gene was firstly built using molecular biology techniques. After determining the success of vector construction by DNA sequencing, 293T cells was transiently transfected with liposome. After it was determined that 293T cells had been successfully transfected with gene expression in S2 by RT-PCR detection, HBV DNA vaccine (pVAX-S2) alone, HBV DNA vaccine combined with different concentrations of naringenin, PEG/pVAX-S2 vaccine were used to immunize C57BL/6 mice, the immunological parameters of anti-HBs IgG, T lymphocyte proliferation, cytokine expression and in vivo killing effect of cytotoxic T lymphocyte (CTL) were detected after14 days of the last immunity.ResultsPVAX-S2, the eukaryotic expression vector containing the HBV pre-S2 and S gene of was successfully constructed. Comparing with the results immunized only with pVAX-S2, the HBV DNA vaccine combined with different concentrations of naringenin (pVAX-S2), PEG/pVAX-S2 could increase the anti-HBs IgG levels of immune mice; after the T-lymphocyte of mixed immune groups was stimulated in vitro by the hepatitis B surface antigen (HBsAg), T cell proliferation was significantly higher than that of control group. In addition, the expression of IL-4 and IFN-γin CD4+ T lymphocytes, and the expression of IFN-γin CD8+ T lymphocytes in mixed immune groups were markedly higher than those in the control group. More importantly, the HBsAg-specific CTL of mixed immune group was significantly higher than that of control group.Conclusions The results show that naringenin can not only enhance the humoral immune response of DNA hepatitis B vaccine, but also enhance the cellular immune response. More importantly, from the therapeutic perspective, the generation of IFN-γin the CD4+ or CD8+ T cells stimulated by the common immune by 1% of naringenin together with the DNA vaccine, will help to remove and control the virus for the chronic infectedperson. It is also found that naringenin can increase the expression of pro-inflammatory factor IL-12, reduce the expression of anti-inflammatory factor IL-35 and TGF-βin T lymphocyte, and activate the downstream signaling molecules of TGF-β, Smad family. Therefore, we speculate that naringenin can provide a micro-environment with a little inhibitory factor, which is more conducive to induce the foreign antigens to produce a stronger immune response. Overall these studies confirme that naringenin has not only enhanced cell-mediated immune response of hepatitis B vaccine and enhanced the therapeutic effect, but also can further explore the mechanism, which will lay a solid foundation in the production practice later. It is also found that the PEG as a carrier transmission system not only can enhance the humoral and cellular immune responses, it can significantly enhance killing activity of in vivo CTL, which will laid a foundation for further research of HBV DNA.
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