【作者】 张传涛；

【导师】 范昕建；

【作者基本信息】 成都中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 乙型肝炎病毒(HBV)感染呈世界性流行,据世界卫生组织报道,全球约20亿人曾感染过HBV,其中3.5亿人为慢性HBV感染者,每年约有100万人死于HBV感染所致的肝衰竭、肝硬化和原发性肝细胞癌。我国属HBV感染高流行区,约有1.2亿病毒携带者,3000万例慢性乙型肝炎患者,每年死于肝炎及其并发症的患者达数十万人,我国HBV感染的直接医疗费用每年约数百亿元。可见HBV感染不仅已经成为严重影响人类健康的医学问题,而且也已经成为加重社会经济负担的社会问题,积极开展防治慢性乙型肝炎研究有重要现实意义,积极开发理想的抗HBV的药物是病毒性肝炎研究领域的热点。目的：研究HH胶囊体内外抗乙型肝炎病毒作用及其部分作用机制。方法：1.将HepG2.2.15随机分组为空白对照组、DMSO组(0.1%)、不同浓度拉米夫定组(300、200、100μg/ml)、不同浓度HH胶囊组(5mg/ml、2.5mg/ml、1.25mg/ml、625μg/ml、312μg/ml、156μg/ml、78μg/ml、39μg/ml、20μg/ml、10μg/ml),分别给予相应的培养液,2天换液一次,分别在第2、4、6天时,用CCK-8检测药物细胞毒性,以找到最大无毒浓度,酶联免疫法(ELISA)检测乙肝病毒表面抗原(HBsAg)和乙肝病毒e抗原(HBeAg),荧光定量聚合酶链反应法(FQ-PCR)检测HBV脱氧核糖核酸(HBVDNA)。2.采用鸭HBV血清制备鸭乙肝模型,随机分为模型组、拉米夫定组(100mg/kg·d)和高(10g/kg·d)、中(5g/kg·d)、低(2.5g/kg·d)剂量HH胶囊组,分别给与相应治疗后,分别在第10、13天时,采用ELISA检测血清中鸭HBV表面抗原(DHBsAg),FQ-PCR检测血清中鸭HBV脱氧核糖核酸(DHBVDNA).3.将HepG2.2.15随机分组为DMSO组和高(312μg/ml)、低(78μg/m)剂量HH胶囊组,分别给予相应的培养液,2天换液一次,第6天时,FQ-PCR法检测细胞内信号转导和转录活化因子(STAT1)、STAT2、干扰素刺激基因因子3(ISGF3)、2′5′-寡腺苷酸合成酶(2′5′-OAS)、RAN依赖蛋白激酶(PKR) mRNA水平,Western Bloting方法检测细胞内OAS、PKR蛋白水平。结果：体外实验表明HH胶囊可以不同程度降低细胞外HBsAg、HBeAg、HBV DNA及细胞内HBV DNA,此作用具有时间和药物浓度依赖性。HH胶囊可以增加细胞内STAT1、STAT2、ISGF3、OAS、PKR的mRNA水平,增强OAS、PKR蛋白水平。体内实验表明HH胶囊可以不同程度降低血清DHBsAg、DHBVDNA,停药后未见病毒复发。结论：HH胶囊体内外均具有抗HBV作用,推测可能与调控JAK-STAT信号通路有关。更多还原

【Abstract】 Hepatitis B Virus (HBV) infection was prevalent worldwide, according to the World Health Organization reporting, about 20 million people worldwide have been infected with HBV, of which 350 million people live with chronic HBV infection, about one million people died each year due to liver failure, cirrhosis and hepatocellular carcinoma with HBV infection. China is a high endemic area of HBV infection, about 120 million people are carriers of hepatitis B virus, of which about 30 million people were chronic hepatitis B patients, up to hundreds of thousands of patients died of hepatitis B and its complications each year,HBV infection leads to direct medical costs of 10 billion yuan each year. So HBV infection not only has become a serious medical problem affecting human health, but also has become a heavier economic burden on the social issues, so being active in prevention and treatment of chronic hepatitis B is important, Actively developing ideal anti-HBV drugs is the focus in the field of viral hepatitis research.Objective:To study the anti-hepatitis B virus effect of HH Capsules and its part of the mechanism in vivo and in vitro.Methods:1. HepG2.2.15 were randomized to the control group, the DMSO group (0.1%), the lamivudine groups with different concentration (300、200、100μg/ml) and the HH capsule groups with different concentration (5mg/ml、2.5mg/ml、1.25mg/ml、625μg/ml、312μg/ml、156μg/ml、78μg/ml、39μg/ml、20μg/ml、10μg/ml), All groups were given the appropriate culture medium, medium was changed once every 2 days, at the time of the day 2,4,6, CCK-8 was used to detect drug toxicity to find the maximum non-toxic concentration, Enzyme Linked Immunosorbent Assay (ELISA) was usede to detect hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg),Fluorescence Quantitative Polymerase Chain Reaction (FQ-PCR) was used to detect hepatitis B virus DNA (HBVDNA).2. Duck hepatitis B model was preparaed by using Duck hepatitis B virus blood,Duck hepatitis B were randomly divided into the model group,the lamivudine group (100mg/kg-d)and the HH capsule groups with the high(10g/kg-d), medium (5g/kg-d) and low (2.5g/kg-d) doses respectively, after giving the appropriate treatment, at the time of the day 10, day 13, ELISA was usede to detect serum DHBsAg, FQ-PCR was used to detect serum DHBVDNA.3. HepG2.2.15 were randomized to the DMSO group, HH capsule groups with High (312μg/ml) and low (78μg/ml) doses, All groups were given the appropriate culture medium, medium was changed once every 2 days, at the time of day 6, FQ-PCR was usede to detect the intracellular Signal Transducers and Activators of Transcription1 (STAT1), STAT2, IFN-stimulated gene factor 3(ISGF3),2’ 5’-Oligoadenylate Synthetase(2’5’-OAS),RAN-dependent protein kinase (PKR) mRNA level, the intracellular protein of OAS, PKR was detected by Western Bloting.Results:In vitro experiment,it shows that, to different degree, HH capsules could reduce the quantity of extracellular HBsAg, HBeAg, HBV DNA and the intracellular HBV DNA in a time and dose-dependent manner.and the HH capsule could increase the intracellular STAT1, STAT2, ISGF3, OAS, PKR mRNA level and enhance the OAS, PKR protein. In vivo experiment,it shows that the HH capsule could reduce the quantity of serum. DHBsAg and DHBV DNA,without virus rebounding after drug withdrawal.Conclusion:HH Capsules has definite anti-HBV effect in vivo and in vitro,likely relating to regulating the JAK-STAT signaling pathway.更多还原