Study On Role Of Toll-like Receptor 7 In Hepatitis B Virus Infection Pathogenesis

BackgroundHepatitis B virus(HBV) infection is the Global health problem. Following initial infection some individuals fail to resolve their infection and thereby become chronic carriers or chronic hepatitis B(CHB), and evern liver cirrhosis or hepatocellular carcinoma(HCC)patients, Serious harm to human health. Chronie HBV infection is associated with phenomenon sueh as T cell hyporesponsiveness or toleranee,lower interferon-αproduction of host,and decreased plasmaeytoid dendritic cells numbers and Impaired fuctions.However,the mechanism on initiation and persistence of HBV infection still remains to be elucidated.Toll like receptors(TLR),a kind of pattern recognition receptors, was discovered in recent years. It playes an important role in the bridge of natural immunity and acquired immunity through pathogen-associated molecular patterns (PAMP) recognition. TLR7 is an important member of the TLR family,which play a central role of antiiviral immuner responses by inducing interferon, interleukin, tumor necrosis factor and other cytokines when binding with its ligand. As the agonist of TLR7,Imiquimod (IMQ/R-847)) and isatoribine((loxribine similar goods) have beening used in skin cancer and chronic hepatitis C treatment. Another ligand of TLR7 Resiquimod (R-848) can significantly inhibit the replication of HBV by inducing IFN-α, IFN-β.TLR7 playes an essential role in antiviral immunity, but in chronic hepatitis B(CHB) patients'peripheral blood mononuclear cells(PBMC),the expression of TLR7 was markedly reduced . However, There's only little study about how the expression and regulation of TLR7 impacted by hepatitis B virus infection in the world now,the role of TLR7 in the pathogenesis and mechanism of hepatitis B infection is uncertain yet.This study in order to described the role of TLR7 in the pathogenesis of hepatitis B infection by researching on the TLR7-mediated anti-HBV effect in vitro, and the expression and regulation signal transduction pathway of TLR7 affected by HBV transfection. To discuss the impairment significance of TLR7 in immune cells after chronic HBV infection by detecting IFN-α,IL-12 content changes which produced by CHB patients'PBMCs on the TLR7-mediated, describe the role and the mechanism of TLR7 in chronic HBV infection.The main aim of this study was to provides credible informations for the study of TLRs-mediated innate immunity and its signal transduction, as well as to provide a theoretical basis for find a new targe for hepatitis B treatment and the development of new drugs.Objective1. To discuss TLR7-mediated anti-HBV effects and the mechanism through stimulating the HepG2.2.15 cell line by TLR7 specific ligand loxoribine in vitro.2. To discuss the effect on expression and regulation signal pathway of TLR7 with the HBV transfected and describe the role of TLR7 in the pathogenesis of hepatitis B ,by detecting the expression of TLR7 after using TLR7 specific ligand loxoribine to stimulate the HepG2 and HepG2.2.15 cell lines which are before and after HBV transfected.3. To study the impairment of TLR7 in immune cells and the significance with chronic HBV infection, by detecting IFN-α, IL-12 content changes which produced by CHB patients'PBMC by the TLR7-mediated, in order to describe the role and the mechanism of TLR7 in chronic HBV infection.Methods1. Selecting HepG2.2.15 cell lines which can synthesis and secrete HBV and HBV marks as a cell model of HBV infection. ELISA and Fluorescent quantificative was used to assay the inhibition of HepG2.2.15 cells secretion HBsAg, HBeAg and the HBV DNA replicationg after TLR7 specific ligand loxoribine stimulated,and dicuss TLR7-mediated anti-HBV effects in vitro.2. Using MTT colorimetric to observe the toxic effects of TLR7 specific ligand loxoribine on HepG2.2.15 cells.3. Flow cytometry was used to detect expression levels and regulation of TLR7 protein of HepG2, HepG2.2.15 cell lines intracellular before and after specifice ligand loxoribine induced.4. ELISA was used to assay IFN-α, IL-12 content changes which are produced by chronic HBV infection patients and healthy human peripheral blood mononuclear cells after stimulated by TLR7 specific ligand loxoribine, evaluation the function of TLR7 after chronic HBV infection.Results1. TLR7 specific ligand Loxoribine could inhibit HBsAg secretion and HBV DNA replication effectively in HepG2.2.15 cells, the inhibition of HBsAg secretion was a dose and time-dependent. There is no effect on HBeAg.2. Low concentration of Loxoribine inhibited the growth of HepG2.2.15 cell at a certain extent ,but as the drug concentration increased, toxic effects of cell growth was apparent.3. HepG2 and HepG2.2.15 cells both express TLR7 intracellular, but the expression of TLR7 in HepG2.2.15 transfected by HBV intracellular significantly lower than HepG2 cells. TLR7 expression in HepG2 and HepG2. 2.15 cells increased a little after loxoribine induction, but there was no statistically significant.4. The level of IFN-αin PBMC culture supernatants of Hepatitis B virus carriers and CHB patients'were significantly lower than healthy people; level of IL-12 in PBMC culture supernatants of CHB patients were significantly higher than healthy people; there was no significant difference of IFN-αand IL-12 level in PBMC culture supernatants between hepatitis B virus carriers and CHB patients .5. After TLR7 specific ligand Loxoribine stimulating, levels of IFN-α,IL-12 in PBMC cultured supernatants of healthy human were significantly increased; levels of IFN-α, IL-12 in PBMC cultured supernatants of hepatitis B virus carriers and CHB patients were also elevated ,but there was no statistically significant. Conclusion1. Specific ligands loxoribine showed remarkably inhibitory effect on anti-HBV effects, its inhibition of HBV probably affected on the stages of viral replication and/or protein synthesis and other links mediated by TLR7.2. HBV infection inhibits TLR7 expression,TLR7 specific ligand Loxoribine induced show a remarkably anti-HBV effect, in other words, decreased expre ssion of TLR7 may involve in the pathogenesis of hepatitis B virus.3. Dysfunction of TLR7 in PBMC of chronic HBV infection, lower production of IFN-α, indiscriminate regulation of IL-12, all of this may be an important reason leading to HBV persistent infection.