甲基斑蝥胺抗乙型肝炎病毒体外实验研究

用２．２．１５细胞株体外抗ＨＢＶ药物模型，对研制的甲基斑蝥胺药物体外抗ＨＢＶ流行性进行评价。应用固相放射免疫测定法检测药物对２．２．１５细胞所分泌的ＨＢｓＡｇ、ＨＢｅＡｇ的抑制效果，应用Ｓｏｕｔｈｅｒｎ转膜杂交法分析药物对ＨＢＶ－ＤＮＡ的区带影响，同时以ＭＴＴ法检测药物细胞毒性。药物对病毒复制指标ＨＢｓＡｇ、ＨＢｅＡｇ的５０％抑制浓度（ＩＤ５０）分别为２．８３ｇ／Ｌ、４．６０ｇ／Ｌ，该药对ＨＢＶ－ＤＮＡ区带无明显影响，药物对细胞的５０％毒性浓度（ＣＤ５０）１０．６０ｇ／Ｌ。药物对ＨＢｓＡｇ、ＨＢｅＡｇ的治疗指数（ＴＩ）＝ＣＤ５０／ＩＤ５０分别为３．７５、２．３０。说明甲基斑蝥胺药物在体外具有一定抗ＨＢＶ活性。     

诺西肽抗乙肝病毒体外实验研究

以ＨｅｐＧ２．２．２．１５细胞株为模型，以其分泌的ＨＢｓＡｇ、ＨＢｅＡｇ、ＨＢＶＤＮＡ及细胞存活率为观察指标，综合评价了诺西肽体外抗ＨＢＶ效果。结果表明：诺西肽对ＨＢｓＡｇ和ＨＢｅＡｇ的５０％抑制浓度ＩＣ５０分别为＜１２．５和４１．６μｇ／ｍｌ，治疗指数（ＴＩ）分别为＞１６和＞４．８。Ｓｏｕｔｈｅｒｎ结果显示，诺西肽在１２．５μｇ／ｍｌ浓度下对细胞内ＨＢＶＤＮＡ的抑制率为２６．４％。     

产前注射乙型肝炎免疫球蛋白阻断乙型肝炎病毒宫内感染

目的：评估乙型肝炎（乙肝）免疫球蛋白（ＨＢＩＧ）阻断ＨＢｓＡｇ阳性孕妇乙肝病毒宫内传播的效果。方法：ＨＢＩＧ组２２例,分２组（各１１例）,均孕ｗｋ２８起予ＨＢＩＧ２００ＩＵ,ｉｍ,Ａ组每３ｗｋ一次,Ｂ组每４ｗｋ一次,均至分娩为止;对照组２３例,不用上述药物。ＨＢＩＧ组每次用药前、分娩前,对照组孕ｗｋ２８及分娩前检测静脉血ＨＢｓＡｇ,ＨＢｅＡｇ,ＨＢＶ＿ＤＮＡ,新生儿出生后检测ＨＢｓＡｇ,ＨＢＶ＿ＤＮＡ。结果：ＨＢＩＧ组孕妇血ＨＢｓＡｇ,ＨＢＶ＿ＤＮＡ明显下降（Ｐ＜０．０５）,ＨＢｅＡｇ无明显变化（Ｐ＞０．０５）。新生儿ＨＢｓＡｇ阳性率明显低于对照组（Ｐ＜０．０５）,２组ＨＢＶ＿ＤＮＡ阳性率差别无显著意义（Ｐ＞０．０５）,未发现孕妇及新生儿不良反应。结论：产前应用ＨＢＩＧ可降低乙肝病毒宫内感染率。     

107例静脉药瘾者丙型肝炎、庚型肝炎病毒重叠感染的研究

目的 了解海洛因静脉药瘾者ＨＧＶ／ＨＣＶ重叠感染状况。方法 用酶联免疫间接法,逆转录套式聚合酶链反应（ＲＴ－ｎＰＣＲ）分别检测１０７例静脉药瘾者抗－ＨＣＶ、ＨＣＶ ＲＮＡ、ＨＧＶ ＲＮＡ。结果 抗－ＨＣＶ（＋）者ＨＧＶ ＲＮＡ（＋）检出率达４８．１５％,而抗－ＨＣＶ（－）者则为２２．６４％,两组比较,差异有极显著意义（Ｘ＾２＝７．５９,Ｐ〈０．０１）。在ＨＧＶ／ＨＣＶ重叠感染者中,单项抗－ＨＣＶ（     

20-7 一种新的对乙型肝炎病毒具有活性的抗病霉药物BMS-200475的代谢情况

ＢＭＳ－２００４７５原是作为抗疱疹病毒药物（ＳＱ－３４６７６）而合成的,对Ｉ型单纯性疱疹病毒（ＨＳＶ－１）、Ⅱ型单纯性疱疹病毒（ＨＳＶ－２）和水痘一带状疱疹病毒具有中度活性,对人巨细胞病毒也有活性,但对ＲＮＡ病毒如人免疫缺陷病毒或流感病毒没有活性。最近发现ＢＭＳ－２００４７５对乙型肝炎病毒（ＨＢＶ）具有高度活性,５０％有效浓度为３．７ｎｍｏｌ／Ｌ,５０％细胞毒浓度为３０μｍｏｌ／Ｌ。用对数生长期细胞检测核苷类药物的胞内代谢,而ＨＢＶ活性的检测则使用停滞期的２．２．１５细胞。在ＭｅｐＧＺ细胞和ＨＢ…     

IL-15表达质粒能增强HIV-1 DNA疫苗诱导的细胞免疫应答

本研究旨在观察白细胞介素１５（ＩＬ－１５）表达质粒能否增强１型人类免疫缺陷症病毒（ ＨＩＶ－ １） ＤＮＡ疫苗诱导的细胞免疫应答及ＩＬ－ １５与 ＩＬ－ ２或 ＩＬ－１２表达质粒是否具有协同作用。 作者分另将ＨＩＶ－１ＤＮＡ 疫苗（ｐＣＭＶ１６０ⅢＢ和 ｐｃＲＥＶ各 ２μｇ）、ＨＩＶ－１ＤＮＡ疫苗和ＩＬ－１５表达质粒 ｐＣＡＧＧＳ－ＩＬ１５（ １、 １０、５０μｇ）、ＨＩＶ－１ＤＮＡ疫苗和 ｐＣＡＧＧＳ－ＩＬ１５同 ＩＬ－２表达质粒 ｐＢＣＭＧＮｅｏ－ｍＩＬ２（１０μｇ）或ＩＬ－１２表达质粒ｐＣＡＧＧＳ－ＩＬ１…     

乙肝散治疗慢性乙肝的临床和实验研究

以清热散毒，健脾化湿两法组成纯中药制剂乙肝散，治疗慢性乙型肝炎４０例，并与齐墩果酸片随机对照，结果发现：治疗组病人除临床症状，体征，肝功能以及蛋白代谢等获明显改善外，乙肝散对乙型肝炎病人ＨＢｅＡｇ有较了的转阴作用，与对照组相比有显著差异（Ｐ〈０．０５），体外试验采用由乙型肝炎病毒（ＨＢＶ）ＤＮＡ克隆转染人肝细胞（ＨｅＰＧ２）的２．２．１５细胞系，对其进行乙肝炎病毒ＨＢｓＡｇ和ＨＢｅＡｇ表达抑制的研     

乙型和丙型肝炎病毒双重感染肝组织病毒标志物的检测

为观察乙型肝炎和丙型肝炎双重感染的尸体肝组织中两种病毒核酸及抗原的分布，对１５例ＨＢＶ与ＨＣＶ双重感染的尸检肝组织用免疫组织化学法检测ＨＢｓＡｇ、ＨＢｃＡｇ和ＨＣＡｇ，Ｄｉｇｏｘｉｇｅｎｉｎ标记的探针原位杂交法分别检测ＨＢＶ ＤＮＡ和ＨＣＶ ＲＮＡ。结果：ＨＢｓＡｇ、ＨＢｃＡｇ、ＨＢＶ ＤＮＡ、ＨＣＡｇ和ＨＣＶ ＲＮＡ的阳性数分别为１２／１５（８０．０％）、１０／１５（６６．７％）、９／１５（６０     

利用2.2.1.5细胞株筛选中草药抗HBV活性的实验研究

为建立体外抗ＨＢＶ 药物筛选的技术,应用乙肝病毒（ＨＢＶ）ＤＮＡ 转染细胞株２－２－１－５ 细胞培养上清中ＨＢｓＡｇ 和ＨＢｅＡｇ 滴度作为药物抗ＨＢＶ 效果的评价指标,对４ 种中药制剂的抗ＨＢＶ 作用进行了评价。同时,应用四甲基偶氮唑盐（ＭＴＴ） 比色分析法检测药物的细胞毒性。结果显示,从中草药中挖掘抗ＨＢＶ 的药物具有较大的潜力,而将ＨＢＶ ＤＮＡ 转染细胞株用于抗ＨＢＶ 药物体外筛选,是一个简单易行的方法。     

聚合酶链反应检测HBVM阳性血清中HBV DNA的临床意义

应用聚合链反应对１４０例乙型肝炎病毒感染标志（ＨＢＶ Ｍ）阳性的慢性肼病患者血清中ＨＢＶ ＤＮＡ进行检测。结果发现：１．ＨＢＶ ＤＮＡ总阳性率为７２．８６％；ＢＨｓＡｇ、ＨＢｅＡｇ和抗ＨＢｃ阳性血清中ＨＢＶＤＮＡ阳性纺分别为７６．５６％、９６．１５％和７４．４２％；ＨＢｓＡｇ阴性血清中ＨＢＶＤＮＡ阳性率为３３．３３％，ＨＢｅＡｇ阴性血清中ＨＢＶＤＮＡ阳性率为５９．０９％。２．各ＨＢＶ感染模式中，Ｈ     

阿地福韦体外抗乙肝病毒的活性

目的：阿地福韦（adefoir dipivoxil,Bis-POM PMEA)是一新的广谱,抗病毒核苷类药物。本实验评价阿地福韦体外抗乙肝病毒（HBV）的活性。方法：应用HBV DNA转染的人肝癌细胞株（Hep G2.2.15),阿地福韦体外抗乙肝病毒（HBV）的活性评价。结果：阿地福韦能显著减少Hep G2.2.15细胞培养上清液中HBsAg,HBV DNA的分泌,Southern印迹法表明：阿地福韦能明显地抑制HBV和制中间体及共价环状闭合DNA。该实验药物浓度对细胞形态,计数及总细胞DNA无明显影响。阿地福韦具有较强的体外抗HBV活性,同时细胞毒性作用不明显。结论：阿地福韦作为新的抗HBV药物,具有其临床应用价值。     

亚硒酸钠对乙型肝炎病毒的体外抑制作用

目的探讨亚硒酸钠在体外对乙型肝炎病毒(HBV)蛋白合成、DNA复制的影响及机制。方法将不同浓度的亚硒酸钠作用于HepG2.2.15细胞系,通过检测细胞培养上清液中乙型肝炎表面抗原(HBsAg)、乙型肝炎e抗原(HBeAg)水平、乙型肝炎核心抗原(HBcAg)和HBV DNA水平来评价HBV复制情况;免疫组织化学检测HepG2.2.15细胞p53蛋白的表达情况。结果亚硒酸钠对HBV复制具有抑制作用,随着亚硒酸钠浓度的升高,对HBsAg和HBeAg的抑制率逐渐上升,但对HBsAg的抑制作用要小于HBeAg。细胞内HBV DNA复制水平也逐渐下降(P<0.01)。p53蛋白的分布也发生了改变。结论亚硒酸钠对HepG2.2.15细胞HBsAg、HBeAg和HBcAg表达、HBV DNA复制均有抑制作用,作用机制与其干扰p53蛋白的表达有关。     

核酸类似物PNA在HepG2．2．15细胞中对乙型肝炎病毒复制的抑制作用

目的评价核酸类似物PNA在HepG2．2．15细胞中对乙型肝炎病毒复制的抑制作用。方法以HepG2．2．15细胞作为细胞模型,拉米夫定作为阳性对照药物。HepG2．2．15细胞于药物处理14d后,收集上清液及细胞。采用ELISA检测上清液中HBsAg和HBeAg含量,HBV荧光定量检测上清液和细胞内HBV DNA水平,CCK-8试剂盒检测药物对细胞的毒性作用。结果核酸类似物PNA与拉米夫定在浓度1．6-1000μmol·L^-1内对HepG2．2．15细胞的毒性均较小。与药物未处理组比较,PNA和拉米夫定均可有效抑制细胞上清HBV DNA,半数抑制浓度（IC50）分别为0．05-0．10μmol·L^-1和0．09-0．18μmol·L^-1,两者之间差异无统计学意义。结论在体外细胞实验中,PNA对细胞的毒性小,与拉米夫定的安全指数相当;PNA对乙型肝炎病毒复制有较强的抑制作用,且具有一定时效量效关系,与拉米夫定的抑制强度相当。     

六月青含药血清对HepG2.2.15细胞HBV复制的抑制作用

目的:研究六月青(LYQ)含药血清对HepG2.2.15细胞乙型肝炎病毒(HBV)复制的抑制作用。方法:采用血清药理学方法,在HBV的体外细胞培养系统(HepG2.2.15细胞)中观察LYQ抗HBV的作用。结果:LYQ含药血清在HepG2.2.15细胞培养中可有效地抑制HBV DNA的复制,其作用呈显著的浓度-效应和时间-效应关系。结论:LYQ在体外能显著抑制HBV,可能是复方六月雪主要活性成分之一。     

Evaluation of hepatitis B virus replication and proteomic analysis of HepG2.2.15 cell line after cyclosporine A treatment

Aim： The effect of cyclosporine A （CsA） on hepatitis B virus （HBV） replication was investigated, and proteomics expression differentiation after CsA treatment was studied in order to provide clues to explore the effect of CsA on HBV replication. Methods： Methyl thiazolyl tetrazolium （MTT） assay was used to evaluate the cytotoxicity of CsA. The HBV replication level in the HBV genomic DNA transfected HepG2.2.15 cell line was determined by an ELISA analysis of hepatitis B surface antigens （HBsAg） and Hepatitis B e antigens （HBeAg） in culture supernatant, while the intracellular HBV DNA replication level was analyzed by slot blot hybridization. Two-dimensional electrophoresis was used to investigate the alteration of protein expression in HepG2.2.15 after CsA treatment in vitro. The differentially-expressed proteins were identified by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry combined with an online database search. Results： CsA was able to inhibit the expression of HBsAg, HBeAg, and HBV DNA replication in vitro in a dose-dependent manner. A proteomics analysis indicated that the expression of 17 proteins changed significantly in the CsA treatment group compared to the control group. Eleven of the 17 proteins were identified, including the overexpression of eukaryotic translation initiation factors （eIF） 3k, otubain 1, 14.3.3 protein, eIF2-1α, eIF5A, and the tyrosine 3/tryptophan 5-mono-oxygenase activation protein in CsA-treated HepG2.2.15 cells. The downregulation of the ferritin light subunit, erythrocyte cytosolic protein of 51 kDa （ECP-51）, stathmin 1/oncoprotein, adenine phosphoribosyl-transferase, and the position of a tumor protein, translationally- controlled 1, was shifted, suggesting it had undergone posttranslational modifications. Conclusion： Our study identified the inhibitory effect of CsA on HBV replication, and found that a group of proteins may be responsible for this inhibitory effect.     

HepG2.2.15细胞中siRNA与拉米夫定抗乙型肝炎病毒复制作用的比较

目的：对HepG2.2.15细胞中siRNA与拉米夫定的抗乙型肝炎病毒（HBV）作用进行比较。方法：构建HBV siRNA的表达载体并转染入HepG2.2.15细胞。分别于48、72、96h收获细胞,与拉米夫定治疗组比较抗HBV作用。用ELISA方法检测HBsAg浓度;HBV DNA水平用实时定量PCR测定;用逆转录PCR检测HBV mRNA水平。结果：拉米夫定能明显降低HBV DNA水平,对HBsAg和mRNA抑制率较低;siRNA能全面降低HBsAg、HBV DNA和mRNA水平（P〈0.05）。结论：HepG2.2.15细胞中,siRNA与拉米夫定的抗病毒作用完全不同,siRNA抗HBV作用明显优于拉米夫定。     

鹅绒委陵菜有效部位(总皂苷)的分离与抗鸭乙肝病毒(DHBV-DNA)作用

目的本文采用大孔吸附树脂(D101型)纯化新工艺技术,对来源于西部药用植物鹅绒委陵菜Potentilla ansserina L.的粗提物进行分离纯化 ,获得具有显著抗肝炎病毒与保肝活性的鹅绒委陵菜有效部位-总皂苷类成分.本文作者重点研究了鹅绒委陵菜有效部位总皂苷类成分体内、体外抗乙型肝炎病毒作用.方法建立以HBV转染的人肝癌细胞系(Hep G2)2.2.15细胞系为体外模型;静脉感染鸭血清DHBVDNA呈强阳性的一日龄北京雏鸭为体内模型;分别观察2.2.15细胞含药培养基中HBsAg和HBeAg及给药治疗后鸭血清中DHBVDNA水平.结果体外实验中,8d 时,各剂量组的鹅绒委陵菜有效部位对HBsAg和HBeAg均有抑制作用.中剂量鹅绒委陵菜有效部位对HBsAg和HBeAg有显著抑制作用(P＜0.01 ).体内实验观察,中剂量和小剂量鹅绒委陵菜有效部位对DHBVDNA有明显抑制作用 (P＜0.05,P＜0.01).结论鹅绒委陵菜有效部位对乙型肝炎病毒具有明显抑制作用,同时为鹅绒委陵菜的研究、开发奠定了良好的基础.     

Protocatechuic aldehyde inhibits hepatitis B virus replication both in vitro and in vivo

Natural compounds provide a large reservoir of potentially active anti-hepatitis B virus (HBV) agents. We examined the direct effects of protocatechuic aldehyde (PA; derived from the Chinese herb, Salvia miltiorrhiza) on HBV replication in HepG2 2.2.15 cell line and duck hepatitis B virus (DHBV) replication in ducklings in vivo. The extracellular HBV DNA, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) concentrations in cell culture medium were determined by quantitative real-time PCR and ELISA, respectively. DHBV in duck serum was analyzed by dot blot. PA appeared to downregulate the secretion of HBsAg and HBeAg as well as the release of HBV DNA from HepG2 2.2.15 in a dose- and time-dependent manner at concentrations between 24 and 48 microg/mL. PA (25, 50, or 100 mg/kg, intraperitoneally, twice daily) also reduced viremia in DHBV-infected ducks. We provide the first evidence that PA, a novel anti-HBV substance derived from traditional Chinese herb S. miltiorrhiza, can efficiently inhibits HBV replication in HepG2 2.2.15 cell line in vitro and inhibit DHBV replication in ducks in vivo. PA therefore warrants further investigation as a potential therapeutic agent for HBV infections.     

Inhibition of hepatitis B virus by D-fraction from Grifola frondosa: synergistic effect of combination with interferon-alpha in HepG2 2.2.15

In this study, D-fraction extracted from Grifola frondosa (GF-D) and its combination with human interferon alpha-2b (IFN) were investigated for the inhibitory effect on hepatitis B virus (HBV) in HepG2 2.2.15 cells (2.2.15 cells). HBV DNA and viral antigens were analyzed by a quantitative real-time polymerase chain reaction and end-point titration in radioimmunoassays, respectively. The results showed that GF-D or IFN alone could inhibit HBV DNA in 2.2.15 cells with the 50% inhibitory concentration (IC50) of 0.59 mg/ml and 1399 IU/ml, respectively. We further investigated the combination of GF-D and IFN for anti-HBV activity and found that they synergistically inhibited HBV replication in 2.2.15 cells. In combination with 0.45 mg/ml GF-D, the apparent IC50 value for IFN was 154 IU/ml. This 9-fold increase in antiviral activity of IFN suggested that GF-D could synergize with IFN. These results indicate that GF-D, in combination with IFN, might provide a potentially effective therapy against chronic HBV infections.     

Cytotoxicity and anti-hepatitis B virus activities of saikosaponins from Bupleurum species

Saikosaponins, the main active constituents of Bupleurum spp., have been shown to possess immunomodulatory, hepatoprotective, anti-tumor and anti-viral activities. In this study, saikosaponins a, c and d were evaluated for cytotoxicity and anti-hepatitis B virus ( HBV) activities. Results showed that, with the exception of saikosaponins a and d, HBV-transfected human hepatoma cells (2.2.15 cells) cultured with saikosaponin c showed a significantly lower level of HBeAg in culture medium. Saikosaponin c also possessed activity in inhibiting HBV DNA replication; this inhibitory effect was not due to the cytotoxicity of saikosaponin c or its effect on 2.2.15 cell proliferation. Although saikosaponin d exhibited cytotoxicity on 2.2.15 cells, it failed to inhibit HBV multiplication. The cytotoxicity of saikosaponin d against HepG2 human hepatocellular carcinoma cells was due to the induction of apoptosis through the activation of caspases 3 and 7, which subsequently resulted in poly-ADP-ribose-polymerase (PARP) cleavage. DNA fragmentation was clearly noted at more than 6 h after HepG2 cells exposure to saikosaponin d. The present study concludes that saikosaponin c exhibits anti-HBV activity and saikosaponin d possesses potent cytotoxicity against human hepatocellular carcinoma cells.