中药白头翁体外抗病毒作用

目的:研究白头翁不同极性的提取物对呼吸道合胞病毒(RSV)的体外抑制作用.并通过进一步分离纯化,为筛选抗RSV的有效单体做准备.方法:预实验分别以水、体积分数为75%乙醇、乙酸乙酯为溶剂进行粗提取药物.分别采用RSV-MA104、单纯疱疹Ⅰ型病毒(HSV-1)-MA104和肠道病毒71型(EV-71)-RD细胞感染模型进行病毒筛选,初步检测其体外具有抗RSV活性.根据预实验结果,再用水提醇沉法对白头翁水总提取物分离纯化,将其上清液和沉淀物分别进行体外抗病毒试验,再用大孔树脂吸附法提取白头翁的16个化学部位进行体外抗病毒试验,通过在显微镜下观察细胞病变效应(CPE),以酶标仪测定中性红染色后的A540值,计算其治疗指数(TI).结果:白头翁的水、乙醇、乙酸乙酯3种总提取物中水总提取物对呼吸道合胞病毒的抑制效果最好;再用水提醇沉法将白头翁水总提取物分离后,沉淀物的治疗指数TI97.01;白头翁大孔树脂水洗脱液2号具有显著的抗RSV活性,治疗指数TI112.99.结论:白头翁水部位具有良好的抗RSV活性.     

菊芋多糖的提取分离及药理作用

目的:研究菊芋多糖的降血糖及抗病毒作用,开发菊芋的药用价值.方法:水提醇沉法提取多糖、Sevag法纯化,以苯酚-硫酸法对菊芋多糖的纯度进行测定;采用四氧嘧啶高糖小鼠造模法造模,以不同剂量给药,测定小鼠的血糖值,观察其活动状况和体质量变化;将纯化后的菊芋多糖进行体外抗病毒实验,通过显微镜观察细胞病变效应(CPE),噻唑蓝(MTT)法染色,酶标仪测定吸光度值,计算治疗指数(TI).结果:经纯化后的菊芋多糖纯度为71.54%,提取率为68.99%,多糖含量较高;经降血糖实验发现,菊芋多糖有良好的降血糖作用;在细胞水平下,通过抗病毒实验得,菊芋多糖对HSV-1、RSV、EV-71病毒均有一定程度的抑制效果,其TI为分别为9.849、51.191、13.826.结论:菊芋多糖具有降血糖和抗病毒的作用,药物安全性好,有较高的开发价值.     

双黄连口服液体外抗单纯疱疹病毒1型的初步探讨

目的探讨双黄连口服液体外抗单纯疱疹病毒1型(HSV-1)的作用.方法选用非洲绿猴肾细胞VERO作为体外培养靶细胞,建立HSV-1感染,用不同剂量的双黄连口服液处理HSV-1感染细胞,以50%组织细胞感染量(TCDI50),细胞病变效应(CPE)为评测指标;采用MTT法,RT-PCR及流式细胞技术作为检测手段并比较药物处理组与HSV-1感染组细胞间病毒表达的差异.结果浓度为标准剂量10%的口服液可明显抑制HSV-1 gD 基因mRNA的转录,选择性抑制HSV-1 DNA的合成;在标准药物剂量0.1%-10%范围内病毒感染组细胞与实验组细胞间凋亡呈现差异性.结论双黄连口服液对体外感染的HSV-1型病毒存在抑制作用,显示出一定的抗病毒活性.     

BV、HSV-1、HSV-2在Vero细胞上增殖的比较研究

为进一步证实1997年本实验室从我国猕猴群中分离的B病毒(BV)与人单纯疱疹病毒1型(HSV-1)、2型(HSV-2)之间的关系,在Vero细胞上对3种病毒的增殖规律及引起的细胞病变进行了比较研究.结果表明新分离BV与HSV-1、HSV-2引起的细胞病变不同,BV主要见细胞融合;病变发展速度不同,BV介于两者之间;对抗BV血清呈不同程度的交叉反应,HSV-1最高仅达70%左右.说明目前国内一直沿用的用HSV-1抗原替代BV抗原检测B病毒抗体的方法,确有漏检的可能.     

几种瑶药的体外抗病毒活性初步研究

对瑶药中蕨类植物药翠云草、半边旗、白毛蛇、海金沙草的提取物的体外抗病毒活性进行初步的筛选.采用四甲基偶氮唑盐比色法(MTF assay)和细胞病变抑制法(CPE reduction assay),对这4种植物药的水提物、醇提物及醇提物的各萃取部位进行体外抗单纯疱疹病毒(HSV-1)、柯萨奇B组3型病毒(Cox B3)的活性测定.结果:这4种植物药对HSV-1或Cox B3都有一定的体外抑制作用,其中翠云草醇提物的乙酸乙酯萃取部位具有较好的体外抗HSV-1和Cox B3活性,半数抑制质量浓度(IC50)分别为12.5和6.25μg/mL,选择性指数(SI)分别为8.3和20;半边旗醇提物的石油醚部位具有较好的体外抗HSV-1活性,IC50为6.25 μg/mL,SI为61.通过对翠云草、半边旗、白毛蛇、海金沙草的抗病毒筛选,发现翠云草的乙酸乙酯部位和半边旗的石油醚部位具有较好的抗病毒活性.     

中药佩兰体外抗病毒有效部位筛选

目的:在细胞水平下,通过抗病毒实验,筛选佩兰最佳提取方法及敏感病毒株,并确定分离纯化效果最佳的大孔吸附树脂型号,为进一步分离非挥发油类抗病毒成分奠定基础.方法:先通过水提法、水提醇沉法、醇提法、醇提水沉法、乙酸乙酯提取法获得粗提物,在细胞水平下,由显微镜观察细胞病变效应(CPE),噻唑蓝(MTT)法染色,酶标仪测定吸光度值,通过比较治疗指数(TI),对肠道EV-71病毒、单纯疱疹Ⅰ型病毒、柯萨奇病毒B5、呼吸道合胞病毒进行体外抗病毒筛选;其次通过大孔吸附树脂分离法,以蒸馏水、体积分数为25%、50%、75%的乙醇作为洗脱剂冲洗树脂柱,筛选分离纯化效果最好的大孔吸附树脂.结果:经初步筛选,水提醇沉法中的沉淀对肠道EV-71病的抑制作用最强,TI为84.631;通过大孔吸附树脂法得,AB-8弱极性大孔吸附树脂对佩兰的分离纯化效果最好,其中体积分数为25%乙醇洗脱部位的T1值可达127.001.结论:体积分数为25%乙醇洗脱部位是佩兰抑制肠道EV-71病毒的最佳有效部位.     

黄芩射干汤主要黄酮类成分与H_7N_9病毒神经氨酸酶的分子对接

目的研究黄芩射干汤主要黄酮类成分与H_7N_9病毒神经氨酸酶(neuraminidase)的分子对接情况。方法检索CNKI数据库,挖掘黄芩射干汤中主要黄酮活性成分,利用AutoDock Vina软件与H_7N_9病毒神经氨酸酶进行分子对接。结果汉黄芩素、黄芩素、木蝴蝶素A等3种黄酮类活性物质与H_7N_9病毒神经氨酸酶结合能最佳,分别为-7.1,-7.2和-7.2 kcal/mol(1 kcal/mol=4.18 kJ/mol)。结论黄芩射干汤主要黄酮类成分具有抑制H_7N_9病毒神经氨酸酶和抗病毒的药理作用,能从多个靶点、多条途径发挥抗病毒感染的作用,有广阔的开发应用前景。     

藏药四臣止咳颗粒剂抗菌抗病毒作用研究

目的:探讨藏药四臣止咳颗粒剂的抗菌、抗病毒作用。方法:采用二倍稀释法、平板转种法测定四臣止咳颗粒剂的最小抑菌浓度(MIC)及最小杀菌浓度(MBC),采用孙氏综合法测定四臣止咳颗粒剂对金黄色葡葡球菌ATCC25925株、流感嗜血杆菌58535株、甲型流行性感冒病毒FM1株、3型腺病毒标准株感染小鼠的半数保护量。结果:四臣止咳颗粒剂对试验所选用的458株临床分离菌株均有一定的抑制和灭活作用,对感染金黄色葡葡球菌ATCC25925株和感染流感嗜血杆菌58535株的小鼠及感染甲型流感病毒FM1株、3型腺病毒标准株的小鼠均有显著的保护作用。结论:四臣止咳颗粒剂具有明显的抗菌抗病毒作用。     

美洲大蠊提取物CII-3体内外抗HSV-2实验研究

目的:研究美洲大蠊提取物CII-3抗HSV-2作用。方法:通过MTT法和CPE法测定美洲大蠊提取物CII-3抗HSV-2活性,计算其TC50、IC50及TI。以雌性小鼠生殖器疱疹为动物模型,在小鼠阴道内注射50μL HSV-2病毒原液,1次/d,连续7 d造模。以ACV为阳性对照,采用灌胃给药治疗10 d。应用ELISA试剂盒检测小鼠血清中TNF-α、IFN-γ、IL-2的水平变化;称量小鼠免疫器官重量,计算脏器指数。结果:美洲大蠊提取物CII-3有抑制HSV-2致细胞病变的作用,其TC50值为190.55μg/m L,IC50值为28.71μg/m L,TI值为6.64μg/m L;美洲大蠊提取物CII-3能够提高小鼠血清中TNF-α、IFN-γ、IL-2的水平,同时能够使小鼠的免疫器官重量增加。结论:美洲大蠊提取物CII-3有抗HSV-2作用,其抗病毒作用可能是通过提高机体免疫功能来实现。     

子宫颈癌致病因子人乳头瘤病毒的发现——2008年度诺贝尔生理学或医学奖成果简介

瑞典卡罗林斯卡(Karolinska)学院诺贝尔奖评审委员会宣布德国科学家哈拉尔德·楚尔豪森(Harald zur Hausen)因发现人乳头瘤病毒(HPV)导致子宫颈癌,与另外两位法国科学家弗朗索瓦丝·巴尔-西诺西(Franoise Barré-Sinoussi)和吕克·蒙塔尼(Luc Montagnier)共享2008年度的诺贝尔生理学或医学奖。楚尔豪森以科学创新的精神和严格的科学实验向当时盛行的"单纯疱疹二型病毒(HSV-2)是子宫颈癌的致病因子"假说发起挑战,经过10多年的研究,于1983、1984年先后在人子宫颈癌活检标本中检测到HPV16、HPV18的基因序列,并成功克隆了HPV16型、HPV18型基因组,揭示HPV16、HPV18是子宫颈癌发病的重要病因。相继又鉴定了多种型别的HPV,提出了HPV家族具有"异质性"。发现HPV16、18的早期基因E6和E7整合于子宫颈癌宿主细胞的基因组内,提出E6和E7基因是诱发子宫颈癌的关键基因。这些重大发现极大推进了HPV研究领域的发展,也为今日HPV预防性疫苗的问世以及新HPV检测技术的不断发展奠定了科学基础。     

Antiviral activities of extracts from Hong Kong seaweeds

We extracted six Hong Kong brown seaweed species with hot water for their antiviral properties. The cytotoxicity and antiviral activity of these extracts were tested by MTT [3-（4,5-dimethylthiazol-2-yl）-2,5-diphenlytetrezolium bromide] method, cytopathic effect reduction assay, and plaque reduction assay. The antiviral effect was further determined by flow cytometric analysis. The results showed that most of these extracts inhibited the propagation of herpes simplex virus types 1 and 2 （HSV-1 and HSV-2） standard strains with very low cytotoxicity to the host cells. The extracts ofHydroclathrus clathratus and Lobophora variegata showed more potential anti-HSV activities than the extracts of the other four seaweeds. They also had moderate antirespiratory syncytial virus （RSV） activities but could not inhibit influenza A virus. Hydroclathrus clathratus was further extracted by diluted acid and alkali and the antiviral effects of the extracts were also detected. The result showed that the hot water extract contained the main carbohydrate components that exhibited the antiviral activities against various strains of HSV, including the acyclovir-resistant strain. HI-3, a compound fractionated from this hot water extract, showed a dose-dependent anti-HSV activity in flow cytometric analysis and plaque reduction assay.     

苦参中钠-葡萄糖协同转运蛋白2抑制活性成分的研究

对一个含400种中草药和民族药的组分库进行活性筛选,结果显示苦参的甲醇提取物具有良好的抑制钠-葡萄糖协同转运蛋白2（SGLT2）的活性.在生物活性导向的分离纯化策略下,运用中压柱色谱和半制备高效制备液相色谱对苦参甲醇提取物的活性成分进行分离纯化,根据理化性质和波谱数据鉴定所分离化合物的结构,并对所得到的化合物进行抑制SGLT2活性测试.从苦参的甲醇提取物中分离得到7个黄酮苷类化合物,鉴定为芒柄花素-7-O--D-吡喃木糖基-(16)--D-吡喃葡萄糖苷（1）、芒柄花素-7-O--D-呋喃芹糖基-(16)--D-吡喃葡萄糖苷（2）、三叶豆紫檀苷（3）、(-)-高丽槐素-3-O--D-吡喃半乳糖苷（4）、6-乙酰三叶豆紫檀苷（5）、罗思菌素（6）、芒柄花苷（7）.除化合物3外,其他化合物均为首次从苦参中分离得到.7个化合物均有抑制SGLT2的活性,其中具有高丽槐素苷元结构的苷类成分3～5具有较好的抑制SGLT2的活性,对SGLT2的抑制活性的IC50范围为2.61～4.47 mol/L.     

金莲花研究现状与展望

金莲花在生药学、药物化学、药理学、药剂学和临床等方面具有很高的应用价值.金莲花主要有效成分为黄酮类,从该黄酮类可分离出牡荆苷、荭草苷等化学物质.黄酮类具有抗菌和抗病毒作用.多种金莲花制剂已用于临床治疗呼吸道和肠道感染.因此,值得对金莲花进行深入研究,以得到具有更强药理活性的有效成分和发现新的药理活性.金莲花制剂可能成为一种有前途的新药.     

Expression of PHGPx in mammalian cells and its antiviral effect against coxsackievirus group B

To express human phospholipid hydroperoxide glutathione peroxidase (PHGPx) in eukaryotic cells and to study its antiviral effect against Coxsackievirus B3m (CVB3m) in vitro, PHGPx cDNA was amplified from a human testis library using specific primers and cloned into expression vector pcDNA3. I/His. Expression of PHGPx was performed in COS-1 cells. The antiviral effect was studied by the treatment of HeLa cells with the recombinant PHGPx. Results showed that the activity of PHGPx expressed in COS-1 cells was 5-fold higher than that in control group, and it inhibited the cytopathic effect on HeLa cells caused by CVB3m. It can be concluded that recombinant PHGPx expressed in COS-1 cells has antiviral effect against CVB3m in vitro.     

An immunologically active chimaeric protein containing herpes simplex virus type 1 glycoprotein D

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) cause both persistent and latent infections, including recurrent cutaneous disease, lethal neonatal disease, central nervous system disease and other clinical syndromes. Modified live vaccines or conventionally prepared subunit vaccines have generally been unsuccessful in the treatment of HSV-1 and HSV-2 infections from the standpoints of safety and efficacy. It has been established that HSV-1 and HSV-2 infectivity may be neutralized in vitro with antisera directed specifically against each of the four major glycoproteins of the virus (gA/gB, gC, gD and gE) and antisera against glycoprotein gD, of either HSV-1 or HSV-2, are capable of neutralizing both HSV-1 and HSV-2 infectivity in vitro and in vivo. We have previously reported on the identification, DNA sequence and expression at low level in Escherichia coli of the gD gene of HSV-1 strain Patton. Here we describe construction of a hybrid gene encoding a chimaeric protein containing HSV-1 gD, bacteriophage lambda Cro and E. coli beta-galactosidase (gD-beta-gal) protein, which is expressed at high level in E. coli. Moreover, the chimaeric protein elicits antibodies in rabbits that not only immunoprecipitate gD from cells infected with HSV-1 and HSV-2 but also neutralize HSV-1 and HSV-2 infectivity in vitro.     

鸡胚尿囊膜法评价板蓝根提取液抗H1N1流感病毒活性作用

 为探讨板蓝根药材、板蓝根颗粒提取液及板蓝根活性组分对流感病毒活性的影响作用,应用鸡胚尿囊膜法评价了板蓝根药材及板蓝根颗粒提取液对流感病毒的预防、治疗和病毒中和作用;评价了板蓝根活性组分对流感病毒的中和应用。研究结果表明,板蓝根药材、板蓝根颗粒提取液及板蓝根活性组分对流感病毒活性具有良好的抑制作用。板蓝根药材提取液对流感病毒具有预防和中和病毒的作用,板蓝根颗粒提取液和板蓝根活性组分具有中和流感病毒的作用。     

An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection

Herpes simplex virus 2 (HSV-2) infection causes significant morbidity and is an important cofactor for the transmission of HIV infection. A microbicide to prevent sexual transmission of HSV-2 would contribute substantially to controlling the spread of HIV and other infections. Because RNA interference (RNAi) provides effective antiviral defence in plants and other organisms, several studies have focused on harnessing RNAi to inhibit viral infection. Here we show that vaginal instillation of small interfering RNAs (siRNAs) targeting HSV-2 protects mice from lethal infection. siRNAs mixed with lipid are efficiently taken up by epithelial and lamina propria cells and silence gene expression in the mouse vagina and ectocervix for at least nine days. Intravaginal application of siRNAs targeting the HSV-2 UL27 and UL29 genes (which encode an envelope glycoprotein and a DNA binding protein, respectively) was well tolerated, did not induce interferon-responsive genes or cause inflammation, and protected mice when administered before and/or after lethal HSV-2 challenge. These results suggest that siRNAs are attractive candidates for the active component of a microbicide designed to prevent viral infection or transmission.     

基于网络药理学探讨金荞麦抗呼吸道合胞病毒作用机制

基于网络药理学筛选金荞麦抗呼吸道合胞病毒的核心作用成分及靶点,并使用芯片数据挖掘和分子对接做初步验证。通过中药系统药理学数据库与分析平台(traditional Chinese medicine systems pharmacology database and analysis platform,TCMSP)筛选核心作用成分,借助SwissTargetPrediction预测核心作用成分靶点,通过GeneCards、GenCLiP 3、NCBI获取呼吸道合胞病毒涉及肺炎靶点。经靶点映射生成交集靶点网络,并通过STRING、DAVID平台构建蛋白质-蛋白质相互作用(Protein-protein interaction,PPI)关系及核心富集通路,获取核心靶点与通路。使用GEO芯片数据挖掘及AutoDock Vina分子对接初步验证核心靶点的有效性。最终通过TCMSP筛选到金荞麦15个成分,经靶点映射发现金荞麦通过45个靶点发挥作用,通过PPI和KEGG(Kyoto encyclopedia of genes and genomes) 通路综合分析可得金荞麦发挥抗呼吸道合胞病毒的作用靶点为AKT1、VEGFA、PTGS2、SRC、EGFR、KDR、STAT3、BCL2等,数据挖掘和分子对接结果与预测基本一致,为进一步深入揭示其作用机制奠定了良好基础。     

芦荟凝集素提取及促免疫活性功能初步研究

分别从3种有经济价值的芦荟品种,即中华芦荟（Aloe vera .ver.chinensis(Haw））,库拉索芦荟（Aloe barbadensis Mill)和木立芦荟（Aloe arborescens Mill)中采用凝胶层析法提取了3种凝集素,Lecl,Lec2和Lec3,经过聚丙烯酰胺凝胶电泳分析其为单一成分的糖蛋白,比较了3种凝集素的糖和蛋白的含量,得出库拉索芦荟凝集素Lec2糖含量最高,木立芦荟凝集素Lec3蛋白含量最高,免疫学实验证明,库拉索芦荟凝集素的凝集效价最高,其次为木立芦荟,中华芦荟,3种凝集素对淋巴细胞转化功能都有明显的促进作用,其中库拉索芦荟凝集素Lec2的促淋巴细胞转化率最高,因此芦荟凝集素在治疗免疫功能低下方面可能有一定的应用价值。     

Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells.

Replication of DNA occurs at discrete sites in eukaryotic cell nuclei, where replication proteins are clustered into large complexes, or 'replicases'. Similarly, viral DNA replication is a highly structured process, notably in herpes simplex virus type-1 (HSV-1; reviewed in ref. 4) in which large globular 'replication compartments' containing the viral replication machinery exist. Replicating cellular DNA redistributes to these compartments upon HSV-1 infection. We have now used antibodies raised against several cellular proteins to detect changes in their subnuclear localization on HSV-1 infection. We found that various proteins involved in cellular DNA replication move to sites of viral DNA synthesis, whereas a selection of non-replication proteins do not. The retinoblastoma protein and p53 (the products of two putative anti-oncogenes) relocate to the same sites as known DNA replication proteins, suggesting that they may be associated with DNA replication complexes in normal, uninfected cells.