【作者】 黄穗；

【导师】 蔡挺；

【作者基本信息】 浙江大学 ， 病原生物学， 2002， 硕士

【摘要】 单纯疱疹病毒(herpes simplex virus，HSV)是人类重要的致病病毒，能引起口唇疱疹、生殖器疱疹、脑炎、内脏器官感染，并与宫颈癌和唇癌有关。人们虽然致力于研制疫苗用于HSV感染的预防，但因HSV的基因为双链DNA，易与宿主DNA发生整合作用，或形成潜伏状态，从而限制了病毒疫苗的发展与应用。筛选有效的抗病毒药物是当务之急。到目前为止，已发现多种化学药物有抗疱疹病毒作用，但往往因为毒性太大或耐药性的产生而限制了它们的临床应用。而祖国的中草药资源非常丰富，有化学药物无法比拟的优点。甘草是最常见的中草药，现代研究证实，甘草酸(glycyrrhizic acid，GA)是甘草中最重要的有效成分之一，抗病毒作用是甘草酸类的主要药理作刚之一。甘草酸的衍生物---甘草甜素(GL)片已正式在临床上用于治疗乙肝病人。日本学者研究证实，甘草酸有直接的抗HBV作用及对肝功能障碍的改善作用。1986年日本学者首次报导了甘草甜素抗爱滋病病毒(HIV)的作用。甘草酸对其他病毒的抑制作用也有一些报道，Utsunomiya等认为甘草甜素的抗流感病毒作用与甘草甜素诱导体内T淋巴细胞产生γ干扰素有关。干扰 浙江人学匹学院颀I：论义I！“学敝体外抗抱疹病亩作用 素（IF）是一类重要的细胞素，它们在同种细胞上具有广谱的抗病毒、 抗细胞分裂、免疫调节等多种生物学活性。2’，5’寡腺昔酸合成酶Q’，5’ OllgO＆dCllylstC SyflthCt8SC，2－SAS）是干扰素抗病毒、抗细胞增殖过程中 的关键酶，该酶可作为一项生化指标用于评价机体 IFN系统的抗病毒作 D 用。 卜一·本实验旨在研究甘草酸抗病毒（单纯抱疹病毒）作用，并进一步了 解甘草酸抗病毒的机理所在。通过此研究有助于明确甘草酸对单纯炮疹 病毒引起的疾病的治疗作用，通过检测甘草酸用药前后2’，r寡腺昔酸 合成酶的变化了解甘草酸抗病毒机理，使甘草酸广泛用于病毒性疾病的 治疗。 对象和方法： 攻击病毒为 HSV，而 HepZ细胞为靶细胞，药物一甘草酸是 SIGMA公司产品，为白色粉木状的甘草酸单铰输，分子量为 82．4， HPLC测定纯度为乃％。先测定病毒毒力；再进行药物细胞毒性实验；药 物抗病毒实验，包括：甘草酸对病毒所致的细胞病变的抑制作用、甘草 酸对病毒侵入细胞的阻断作用、甘草酸对病毒的直接抑制作用、相同浓 度甘草酸对不同浓度病毒的抗病毒作用；给小鼠注射不同浓度甘草酸， 检测注射diJ与注射后 6小时、互二小时、24 ／h时、48小时 2’，5’寡腺昔酸 合成酶的变化。 结果： l、药物细胞毒性实验 r 甘草酸对HepZ细胞的最大无毒浓度为＊sing／ml，而且使用无毒 浓度甘草酸的细胞生长得比对照组更好，维持时间更长，说明甘草酸有 促进细胞生长的作用。 2、药物抗病。8实验： 2 浙江人学医学院灿卜论义 仕竿胶休外抗抱疹刑详作用 2＊ 甘草酸对病毒所致的细胞病变的抑制作用 将甘草酸连续 2倍稀释做抗病毒实验表明在 0．5到2smg／ml间， 有明显的细胞病变抑制作用，说明甘草酸对HSVI的复制有抑制作用。 N Reed－Muench法计算细胞病变抑制率，ED扣为 0．574mg／ml。 2．二 甘草酸对病毒侵入细胞的阻断作用 用 25、l＊、0．5、0．25、0．128、0．064mg／ml白甘草酸对病毒感染 的预防作用表明，2．5、1．0和 0．sing／ml浓度组可延缓 HSV引起的细 胞病变，与病毒对照组有明显差异；而V草酸浓度（0．25 mg／ml则无明 显阻断病毒侵入细胞的作用。提示大剂量的甘草酸才有预防HSVI 感 染的作用。＆ Reed－Muench ik计算细胞病变抑制率，ED扣为 1．89mg／ml。 2．3 对病毒的直接抑制作用 将以上6个浓度的甘草酸与HSVI悬液混合、经37℃作用6、12 小时，检测甘草酸对HSVI感染的阻断作用。结果表明，二者对HSVI 感染无明显减弱，细胞病变程度与病毒对照组无显著差异。 2．4 相同浓度甘草酸对不同浓度病毒的抗病毒作用 选用 lffig／ffil甘草酸进行对 100TCID50f00 TCIDS卜 1000 TCIDS。的 HSVI抗病毒研究，包括以上三过程。结更多还原

【Abstract】 Herpes simplex virus (HSV) is an important pathogen which can cause herpes labials, Herpes genitalia, encephalitisand splanchnic organ infection, and related with cervix or labio ’27’carcinoma. Great effort spent in preparing vaccines to prevent HSV infection, but as the DNA virus with double strains, and its tendency to ingrated with host DNA or latent infection, so the limitation restriced the development and usage of vaccine, and it is urgent to screen the effective anti-HSV drug. To date, several chemical compounds have been found the anti-HSV function, but often accompanied with not allowable toxicity or the production of drug resistant strain. Chinese medicinal herbs can avoid these limitations in some extents. Glycyrrhizin, as the most seen herb, recent research confirmed that glycyrrhizic acid, GA was the important component in glycyrrhizin, and presented its anti virus function in pharmacology. GA derivative GL had been used in clinical treating hepatitis formally. Japan scholars revealeddirectly anti-HBV and improved the function of liver. And in 1986, they first reported GL also had the effect in resisting HIV. As for the other virus, Utsunomiya and colleagues suggested the function of GL anti influenza virus may by inducing Y -IFN increase through the excretion of T cell. IFN, being as one of the most important cytokines , showed several biological activity such as widely anti-virus, resisting cell division, regulating immune response on cell background. And in IFN pathway, 2’, 5’-Oligoadenylate synthetase (2-5AS) was the key enzyme, the change of this enzyme can be one biochemical marker to evaluate the effect of endogenous IFN.The aim of this study is to confirm if GL anti-HSV in vitro, and its possible mechanism of resisting virus. Through these steps, we may learn if GL can cure the diseases caused by HSV, and if involved with IFN system, which could help us to deal with mostly virus in reality.Object and Method:HSV-1 strain as the attacking virus, Hep-2 cell as the target; glycyrrhizin was bought from SIGMA Co, MW 821.4,HPLC purity75%. Determine the virulence first, then proceed cytotoxicity of the drug, at last we design the antiviral experiment, which includes: the function of glycyrrhizin for suppressing CPE phenotype; blockage virus entry by glycyrrhizin; direct inhibition of glycyrrhizin; and the function of the same dosage glycyrrhizin to the varying concentration of virus. Inoculating the different dosage glycyrrhizin into mouse, to investigate the change of 2-5AS in a series of time point: before injection, 6h, 12h, 24h, 48h after injection.Result:1 .Cytotoxicity of glycyrrhizin:The maximum no cytotoxicity dosage of glycyrrhizinto Hep-2 cell was 2.5mg/ml, and cell grew better, maintained longer in no cytotoxicity group compare with the control. This phenomenon represent glycyrrhizin has the function to facilitate cell growth.2. Antiviral experiment of glycyrrhizin:2.1 CPE inhibition experiment by glycyrrhizinSeries dilution of glycyrrhizin in 2-fold, then in the range of 0.5-2.5 mg/ml, glycyrrhizin manifested distinct inhibition of CPE. Computed its rate by Reed-Muench method, ED.sowas 0.574mg/mlo2.2 Blockage virus entry of glycyrrhizinEmploying 2.5,1.0,0.5,0.25,0.128,0.064mg/ml glycyrrhizin to prevent the infection, the group of 2.5orl.O or0.5mg/ml could postpone the CPE phenotype, which was significantly difference in virus control; but less than 0.25 mg/ml group had no obvious effect. This situation disclosed glycyrrhizin preventing HSV infection in somewhat high dosage Computed CPE repression rate by Reed-Muench method, EDsowas 1.89mg/mlo2.3 Direct inhibition of glycyrrhizinTaking the six dosage glycyrrhizin listed above mixtured with HSV-1, then incubated in 37癈 6-12h, arranging the virus group as control, the result indicated though after 6 or 12h interaction, there was no obvious weaken of HSV infection.2.4 The function of the same dosage glycyrrhizin to the varying concentration of virusUsing 1更多还原