The Establishment And Evaluation Of In Vitro Drug-screening System For Anti-HBV Drugs And The Analysis Of Anti-HBV Effect Of Green Tea Extract

Hepatitis B virus (HBV) infection causes major public health problemsworldwide. Present treatment strategies for HBV infections are not satisfactory andthe majority clinical limitation of current antiviral drugs for HBV, such as lamivudineis causing rapid emergence of drug-resistant viral strains during the prolongedtherapeutic treatment. Therefore, it is urgent and necessary to explore novel anti-HBVdrugs by using good model and reliable detection methods. In this dissertation, wedynamically analyzed two HBV expression cell lines. A quantative real-time PCRmethod for detecting HBV covalent closed circular DNA (cccDNA) was set up. Withenzyme linked immuno-absorbent assay (ELISA) and real-time polymerase chainreaction (PCR), we validated the inhibitory effects of green tea extract andrecombinant MAP30 against HBV.Chapter 1 was a general introduction. It contained an introduction of HBV, pathological mechanism and treatment of Hepatitis B, the significance of detectingHBV cccDNA, the drug screening models of HBV, and vegetal antiviral factors, suchas green tea extract and MAP30. The aim, contents and significance of this thesiswere also presented.Chapter 2 described the establishment of a method for detection of HBV cccDNA with real-time PCR. Using a pair of primers spaning two gaps of HBVgenome, Eva green fluorescent dye and a standard plasmid, the HBV cccDNAs ofsera samples and HBV expression cell lines were detected. Results showed that themethod was reliable and sensitive.In chapter 3, two cell lines, HepG2-N10and HepG2 2.2.15, which could stablyexpress HBV were analyzed dynamically. With ELISA and real-time PCR, the HBVantigens, intracellular replicative intermediates, extracelluar HBV DNA and nuclearcccDNA were detected. Results showed that HepG2-N10 grew quicker than HepG22.2.15, and secreted more antigens and virons. It indicated that the HepG2-N10 cellline has advantages for drug screening and can be used to complement other currentlyused in vitro antiviral model systems.In chapter 4, the antiviral effect of green tea extract (GTE) against HBV inHepG2-N10 cells was examined. Quantitative real-time PCR was used for thedetermination of extracellular HBV DNA, intracellular replicative intermediates andnuclear cccDNA of HepG2-N10 cells before and after GTE treatment. The expressionof viral antigens, Hepatitis B s Antigen (HBsAg) and Hepatitis e Antigen (HBeAg)were determined by ELISA. HBV mRNAs were analyzed by RT-PCR. Resultsindicated that the majority components of green tea extract polyphenols could inhibitthe production of HBV.In chapter 5, the gene of a bitter melon antiviral protein (MAP 30) was clonedand expressed in E. coli. The purified recombinant MAP 30 showed inhibitory effectsto HepG2 cancer cell, Sphaerotheca fuliginea and staphylococcus aureus. WithHepG2-N10, the anti-HBV activity of MAP30 was analyzed. The inhibition rates toHBsAg and HBeAg were 12.9ï¼…and 26.7ï¼…respectively.