【作者】 胡纯彰；

【导师】 张奉学；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2010， 硕士

【摘要】 由乙型肝炎病毒引起的肝炎是一种严重危害人体健康的病毒性传染病。据世界卫生组织报道,全球有超过20亿人为乙型肝炎病毒携带者,其中约3亿人为慢性终身感染者。而我国的乙肝患者就约占了一半,直到2008年我国乙肝表面抗原携带者约有9300万人。于此同时每年仍约有200万新发肝炎病人,其中乙型肝炎占20%。与非乙肝病毒携带者相比,慢性肝炎感染患者面临较高的肝硬化或癌变的风险。目前在尚未找到能彻底根治乙肝的方法同时,由于近年HBV免疫疫苗、各种抗病毒药物的应用而导致HBV变异株的出现,使HBeAg阴性慢性乙型病毒性肝炎患者(CHBV)增多。而在最新的科学研究中,研究人员发现在感染病毒的肝细胞核内存在的病毒共价闭合环状DNA (covalently closed circular DNA, cccDNA),是病毒持续感染久治不愈的重要原因。因此科学研究者在研制不同类型的临床治疗药物的项目上着重研究药物对乙肝病毒cccDNA的影响,希望能够找到直接可以清除病毒cccDNA的药物,让乙肝病人痊愈成为现实。HBV复制大约分别四个阶段：(1)乙肝病毒颗粒首先与细胞膜上的受体羧肽酶D结合,脱去外膜进入细胞内；(2)在经衣壳蛋白的磷酸化作用后脱去衣壳,乙肝病毒进入细胞内,并在细胞内转变成超螺旋DNA结构(cccDNA)。超螺旋DNA是HBV的转录模板,它可转录mRNA,也是造成乙肝病毒无法清除的重要原因之一；(3)在mRNA指导作用下病毒先合成前基因RNA,并在细胞核外被衣壳包裹,然后再以前基因RNA为模板,在细胞核外经逆转录合成乙肝病毒DNA负链,随后合成乙肝病毒正链；(4)衣壳中的乙肝病毒由膜包裹后分泌到细胞外以完整的病毒颗粒进入到其它细胞,也可重新脱去衣壳进入细胞核内,以维持细胞核内病毒超螺旋DNA(cccDNA)的数量。因此每个病毒复制的步骤都是研究抗乙肝病毒的关键,只要阻断病毒的复制过程就可以有效地抑制病毒的增殖。经过大量的研究,乙肝病毒对肝细胞并无直接致病性。相反由乙肝病毒所引发的肝细胞损害的机制主要是由细胞免疫反应所引起的。在HBV感染过程中,先由单核巨噬细胞摄取病毒抗原并加工,然后提呈给Th细胞。然后在单核巨噬细胞释放的白细胞介素1(IL-1)协助下,促使Th细胞活化增殖而释放白细胞介素2(IL-2)。IL-2刺激已被HBV抗原致敏的Tc细胞发生克隆性增殖,形成大量效应性T细胞,攻击受HBV感染的肝细胞,最后导致肝细胞变性坏死。目前认为, Tc细胞攻击靶细胞需依赖所谓“双识别”,即只有表达靶抗原和Ⅰ类MHC抗原的肝细胞,才可能被Tc细胞识别、攻击和破坏。而肝细胞凋亡、HBV感染等多种因素所致的肝细胞损害,其最终表现也是肝细胞死亡。本实验以乙肝病毒感染机制、慢性乙肝与cccDNA关系的研究为基础,研究实验药物泽泻醇B乙酸酯对乙肝病毒HBV的抑制作用及其作用机制。泽泻为泽泻科植物泽泻的块茎,性味甘、淡、寒,归肾、膀胱经,是利水渗湿用药,主要用于小便不利、水肿胀满、泄泻、尿少、痰饮、眩晕、热淋涩痛等症。泽泻的化学成分比较复杂,而泽泻醇B乙酸酯属于三萜化类合物。在有关泽泻醇B乙酸酯的研究中,学者们发现泽泻醇B乙酸酯具有多种生物活性：(1)诱导多种肿瘤细胞凋亡；(2)逆转P糖蛋白过表达癌细胞的多药耐药性,提高细胞对抗肿瘤药物的敏感性；(3)抑制抗体过敏性反应；(4)抑制细胞中氮氧化物(NO)的合成反应；(5)抑制乙肝病毒抗原活性,等等。因此泽泻醇B乙酸酯在抗病毒方面具有很大的潜力。在本实验过程中,通过体外、体内实验平行检测泽泻醇B对病毒DNA的影响、对病毒cccDNA的影响从而探索泽泻醇B乙酸酯抗乙肝病毒的机制。在实验中,泽泻醇B乙酸酯在2.215细胞和鸭胚肝细胞上的对病毒的抑制作用并不一致。在2.215细胞上,泽泻醇B乙酸酯的TC50为23.71mmol/L,而在鸭胚肝细胞上的TC50为25.62mmol/L。泽泻醇B乙酸酯在HepG2.215细胞上对病毒两抗分泌的治疗指数分别为1.15、0.74,提示在HepG2.215上泽泻醇B乙酸酯对乙型肝炎病毒表面抗原分泌的抑制作用是在对细胞的毒性作用基础上发挥的,而对病毒的e抗原则无抑制作用。在先天DHBV感染鸭胚原代肝细胞上,泽泻醇B乙酸酯对病毒DNA的治疗指数可达到4.08,则显示出较好的抗病毒作用。在体内实验中,泽泻醇B乙酸酯对病毒的DNA和肝组织中的cccDNA也有一定的抑制作用。通过本实验的初步结果间接提示了泽泻醇B乙酸酯需要经过体内代谢的活化作用才能发挥药物的抗病毒作用。更多还原

【Abstract】 Hepatitis is a kind of serious infectious virusis caused by Hepatitis B virus. According to the reports from WTO, over 2 billion people are HBV carriers, and more than 300 million are chronic infected. About half of the HBV infected people are in our country. The number of the HBsAg carriers is 9.3 million till 2008 in China and increase with 2 million every year. Compared with those non-HBV carriers, the HBV carries faced with much higher risk of liver cancer and cirrhosis. With the therapy of HBV vaccine and other anti-HBV medicines, which makes the HBV virus variating quickly, increases more and more chronic HBV infected patients. Scientists find out that the cccDNA of the HBV virus exits in the liver is the key to the chronic infection of HBV. So people put more energy in seeking for the medicine witch can erase viral cccDNA in liver, and make it possible to heal the HBV carriers completely.The HBV replication includes four classes:first, the virus exuviates adventitia and enters into the infected hepatocytes with phosphorylase, the virus draws off the capsid protein and turn to the structure of cccDNA. cccDNA is the HBV replication template, and it also can transcript to mRNA. Then the virus integrates pre-RNA with the instruction of mRNA, and envelops with capsid in extranuclear. The virus reverses transcripts to negative DNA chain with the pre-RNA as template, and synthesizes to positive DNA chain. At last the complete virus which has infectious ability is secreted to extranculear to maintain the numbers of cccDNA. So blocking off the every step of viral replication is the key to heal hepatitis B disease.In a great deal of researches, scientists find that the liver damage is caused by cell-mediated immunity but not the HBV virus itself. In the course of HBV infection, the monocyte-macrophage cells incepts and processes the viral antigen, then submits to the Th cell. With the IL-1 secreted from macrophage, the Th-cells are activated to multiplication and release IL-2. Then the sensitized Tc cells proliferate and turn into effect T cells. The effect T cells attack the hepatocytes affected by HBV virus, which lead to the liver damage. The scientists think the hepatocytes which can express target antigen and type 1 MHC antigen can be attacked by the Tc cells. And the apoptosis of cells and the viral infection are also the reasons of the damage-too.My experiment is based on the mechanism of the HBV infection and the relationship between the chronic hepatitis B and cccDNA to describe the anti-HBV effect of Acetate alisol B. Acetate alison B is isolate from Radix Astragliz. In traditional Chinese medicine, Radix Astragliz attributes to sweet, light, cold, owned by the kidney and bladder, used in diuresis.And the compounds of Radix Astragliz is very complicated. The Acetate alisol B attributes to triterpenoid compounds of classes. It has many activated functions, such as inducing tumor apoptosis, enhancing the sensitive of the cells to the anti-tumor medicine, inhibiting hypersensitivity and the synthesis of NO, restraining HBV virus. So Acetate alisol B is very potential in anti-HBV medicine research.We detect the viral DNA replication, and the presence of viral cccDNA in vivo and in vitro synchronously. In the assay, we found that different results showed in different cell models. On the HepG2.215 cell model, the TC50 of Acetate alisol B was 23.71mM, and the therapy index (TI) of HBsAg and HBeAg were 1.15,0.74 respectively, which showed that the Acetate Aliso B had not anti-HBV effect on HepG2.215 cells. But strangely, the data on primary duck hepatocyte cells showed that the Acetate Aliso B had a great anti-HBV effect on it. The TI of Acetate also B on restrain DHBV DNA was 4.08. The anti-HBV effect of Acetate alisol B is not apparent in HepG2.215 cells, but shows preliminary effect in vivo. So we surmise the Acetate alisol B has the effect to restrain HBV after metabolism in liver.更多还原