蒙药乙肝1号对HBV转染细胞表达功能的影响

目的:探讨蒙药乙肝1号抗HBV药理作用。方法:采用HBV的2.2.15细胞系作为模型,观察药物对2.2.15细胞分泌HBsAg和HBeAg的抑制效果。结果:蒙药乙肝1号30mg/ml浓度对细胞有毒,最大耐受药物浓度20mg/ml时对HBsAg抑制率为57.81％,半数有效剂量(IC_(50)为5.54±3.44mg/ml,治疗指数TI为5.42±3.42;对HBeAg的抑制率为71.64％,半数有效剂量(IC_(50))为3.69±0.01mg/ml,治疗指数(TI)为6.41±0.03mg/ml。结论:提示蒙药乙肝1号煎剂在体外有一定的抗HBV作用。     

肝舒胶囊在体外细胞培养中抗HBV活性的研究

目的研究肝舒胶囊在2.2.15细胞培养中抗乙型肝炎病毒(HBV)作用。方法用2.2.15细胞体外培养.对肝舒胶囊抗HBV活性进行评价。结果肝舒胶囊5g／L加药后4d对2.2.15细胞HBsAg和HBeAg的抑制率为51.16％和74.83％,在加药后第8天抑制作用达高峰,对HBsAg和HBeAg的抑制率分别为75.72％和80.02％。药物作用12d时．其治疗指数可达7.73和5.39。结论肝舒胶囊在体外细胞培养中对HBsAg和HBeAg的分泌均有较好的抑制作用。     

愈肝胶囊对2.2.15细胞分泌HBsAg、HBeAg的抑制作用

目的：观察愈肝胶囊对HBV转染细胞表达功能的影响。方法：本研究以乙型肝炎病毒(HBV)基因转染的人肝癌细胞系2．2．15细胞作为筛选抗HBV药物的细胞模型，观察愈肝胶囊对2．2.15细胞分泌HBsAg、HBeAg的影响；并以四甲基氮唑蓝(MTT)比色法检测药物对细胞的毒性。结果：愈肝胶囊对2．2．15细胞分泌HBsAg和HBeAg有显著抑制作用。半数中毒剂量(TC50)为1．79mg／ml，对2．2．15细胞分泌HBsAg和HBeAg抑制率分别为70．81％，83．96％；半数有效剂量(IC50)HBsAg为0．86，HBeAg为0．13。治疗指数(TI)分别为HBsAg2．08，HBeAg13．76。结论：愈肝胶囊在体外有一定的抗HBV作用。     

黄芩甙对HBsAg和HBeAg的体外抑制作用

目的：观察黄芩甙(Baicalin,Bai)对2．2．15细胞分泌HBsAg和HBeAg的影响，探讨黄芩甙的体外抗HBV作用。方法：通过四甲基偶氮唑盐(MTT)法观察黄芩甙对2．2．15细胞的影响，采用ELlSA法检测分析黄芩甙对2．2．15细胞分泌m6Ag、HBeAg的抑制作用。结果：黄芩甙在2．2．15细胞上半数毒性浓度大于15mg／ml，在该浓度下对HBsAg、HBeAg的抑制率分别达100％、88％以上，在黄芩甙浓度为0．94～15mg／ml时，对2．2．15细胞分泌HBsAg、HBeAg的抑制率高于70％，治疗指数分别为65．22和21．74。结论：黄芩甙有显著的体外抑制HBV作用，属低毒有效药物，具有潜在的抗HBV开发前景。     

人干扰素-α基因转移、表达及其抗乙型肝炎病毒的实验研究

目的研究人干扰素-α(hIFN-α)基因的转移、表达及其抗乙型肝炎病毒的作用.方法利用聚合酶链反应(PCR)技术和基因重组技术,体外扩增hIFN-α基因并构建重组表达hIFN-α基因的逆转录病毒表达载体(pDOR-IFN-α),用NIH 3T3细胞扩增法测定其假病毒颗粒滴度,用染料吸收法检测hIFN-α活性;将假病毒颗粒感染2.2.15细胞,用固相放免法检测其上清HBsAg和HBeAg,用斑点杂交法结合计算机扫描检测其细胞HBV DNA密度.结果PCR克隆出648 bphIFN-α全基因;假病毒颗粒滴度为1×106CFU/ml;hIFN-α生物活性为239 IU/ml/1×106/48 h;对HBsAg的抑制率随培养时间(2、4、6和8 d)的延长而逐渐增高,抑制率分别为33%、55%、55%和62%,而对HBeAg的抑制则在转染后的第2天抑制率最高,以后随培养时间延长而逐渐下降,其抑制率分别为67.7%、40%、19.5%和25.3%.而对照组对HBsAg第2、4、6、8天的抑制率分别为0%、10.8%、0%和13%,对HBeAg的抑制率分别为0%、0%、0%和1.2%.而且对HBV DNA亦有明显的抑制作用,抑制率达66%;而对照组(空白载体)抑制作用不明显.结论hIFN-α基因可以成功地转移和表达,并有抑制HBV复制和表达的作用.     

氧化苦参碱抗乙型肝炎病毒的体外实验研究

目的：观察氧化苦参碱体外抗乙型肝炎病毒（HBV）的活性。方法：以HepG2－2．2．15细胞株为模型, 用微粒酶免疫测定技术(MEIA）检测细胞培养上清液中HBsAg、HBeAg含量,用bDNA信号可扩增法定量检测细胞内核心颗粒HBV DNA的变化,以MTT比色法观察药物的细胞毒性。结果：氧化苦参碱2000μg/ml时对HBsAg、HBeAg的抑制率分别达40．57％、48．27％;浓度为100－200μg/ml时,能明显降低2．2．15细胞浆核心颗粒HBV DNA水平;无明显细胞毒性作用。结论：体外细胞培养表明,氧化苦参碱具有直接抗HBV活性。     

利用体外细胞培养系统筛选抗HBV活性中草药的初步研究

利用HBV的体外细胞培养系统(2215细胞系)建立了体外抗HBV药物筛选的技术常规,并以此对8种中草药注射液的抗HBV活性进行了初步观察。结果显示:板蓝根对2215细胞HBsAg和HBeAg分泌的抑制率分别在22.27%～53.74%和14.54%～     

复方六月雪体外抗HBV的实验研究

目的观察复方六月雪(CLYX)体外抗乙型肝炎病毒(HBV)的作用。方法在最大无毒浓度(TC0)基础上观察不同浓度药物作用于HepG2.2.15细胞,分别在第4天和8天收集细胞培养上清液,采用实时荧光定量PCR法检测上清液HBV DNA的含量,采用ELISA法测定上清液HBsAg和HBeAg的滴度。结果无毒浓度的复方六月雪在HepG2.2.15细胞培养中可有效地抑制细胞HBV DNA的复制及HBsAg和HBeAg的分泌。结论CLYX在体外有显著的抗HBV的作用。     

不同修饰的左氧氟沙星体外抗乙型肝炎病毒的作用及其作用机理的研究

目的本文用2.2.15细胞研究不同修饰的左氧氟沙星(磺酸左氧氟沙星、左氧氟沙星、乳酸左氧氟沙星)体外抗乙型肝炎病毒的作用及其作用机理。方法以HBsAg、HBeAg及细胞存活率为观察指标,综合评价不同修饰的左氧氟沙星体外抗HBV效果,进一步用荧光定量PCR方法测细胞内HBV DNA、HBV RNA及细胞上清HBV DNA的拷贝数,推测药物体外抗乙型肝炎病毒的作用机理。结果结果提示磺酸左氧氟沙星对HBsAg和HBeAg的ID_(50)分别为63.1μg/ml及81.3μg/ml,CD_(50)为182.0μg/ml,TI>2。磺酸左氧氟沙星对细胞内、外HBVDNA及细胞内HBV RNA均有明显的剂量依赖型抑制作用(P<0.05)。结论磺酸左氧氟沙星有明显的抗HBV作用,对HBV抑制位点可能在HBV DNA水平。     

低密度cDNA Macroarray对干扰素α抗病毒蛋白的筛选

目的 基于低密度cDNA Macoarray技术筛选出差异表达的干扰素(IFN)α抗病毒基因,以探讨IFN α抗病毒蛋白的表达与HBV复制的关系. 方法 以一定浓度的IFN α处理肝胚瘤细胞株HepG2和HepG2.2.15细胞6h,用cDNA Macroarray分析比较两细胞株IFN α抗病毒基因表达谱,并筛选出差异表达的IFNα抗病毒基因.将表达HBV核心蛋白(HBc)的质粒pHBc-EGFP转染HepG2细胞,RT-PCR法分析HBc对IFN α抗病毒基因表达的影响.将表达抗黏病毒A蛋白(MxA)的表达质粒pcDNA3.1-Flag-MxA转染HepG2.2.15,以酶联免疫吸附试验、Dot blot、Southern blot等方法分别检测HepG2.2.15细胞表达释放的HBsAg与HBeAg、细胞外HBV DNA和细胞内HBV DNA复制中间体(松弛环状DNA、双股线性DNA),以判断HBV复制情况.两组间数据比较采用t检验,组间不同时间点数据比较采用单因素方差分析.结果 cDNA Macroarray分析显示HepG2和HepG2.2.15细胞的抗病毒基因表达谱具有差异性:IFNa抗病毒基因中干扰素诱导跨膜蛋白(IFITM)1、IFITM2、IFITM3、RING4等在HepG2.2.15细胞的表达被部分抑制,而重要的抗病毒蛋白MxA表达被完全抑制.HBc转染组细胞中MxA mRNA表达的相对水平为0.31±0.05,低于空白对照组的0.74±0.04,差异有统计学意义,P＜0.05.MxA蛋白转染HepG2.2.15细胞48、72 h后,MxA转染组细胞上清液中HBsAg的S/CO值分别为1.42+0.21和1.58±0.18,HBeAg的S/CO值为1.44±0.14和2.28±0.24,而空白对照组细胞上清液中HBsAg的S/CO值为1.92±0.19和2.79±0.25,HBeAg的S/CO值为2.31±0.46和3.37±0.29,两组细胞上清液中HBV抗原的S/CO值差异均有统计学意义,P值均＜0.05.细胞外HBV DNA、胞内HBV复制中间体DNA均无明显变化.结论 HBV及其抗原成分的复制和表达影响着IFNα抗病毒蛋白的表达;HBV通过抑制IFN α抗病毒蛋白的表达而发挥拮抗IFNα的抗病毒活性.     

胸腺肽α1、α干扰素和拉米夫定联合治疗HBV慢性携带者的临床研究

观察胸腺肽α1、α干扰素和拉米夫定联合治疗HBV慢性携带者的临床疗效。选择HBsAg、HBeAg、抗- HBc、HBV DNA阳性,肝功能正常的HBV慢性感染者72例,分为联合治疗组42例:拉米夫定0．1／d,口服,疗程52 周;胸腺肽α1 1．6mg／次,皮下注射,2次／周,连续26周;α干扰素500万单位／日,肌注,疗程26周。单用拉米夫定组 30例:剂量、疗程同联合治疗组。联合治疗组HBV DNA阴转率在26周时76．2％,52周时81．0%,拉米夫定组为 50．0％和66．7％。联合治疗组HBeAg阴转率26周时33．3％,52周时38．1％,拉米夫定组为10％和13．3％。HBeAg 血清转换率两组无统计学差异。联合治疗对HBV DNA和HBeAg的抑制作用显著优于单用药。     

Synergistic effect of a novel oxymatrine-baicalin combination against hepatitis B virus replication, α smooth muscle actin expression and type I collagen synthesis in vitro

AIM: To study the effect of oxymatrine-baicalin combination (OB) against HBV replication in 2.2.15 cells and alpha smooth muscle actin (alpha SMA) expression, type I, collagen synthesis in HSC-T6 cells. METHODS: The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to beta actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by beta actin. RESULTS: In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group (HBsAg, P = 0.043; HBeAg, P = 0.026; respectively); HBV DNA level in the OB group was significantly lower than that in the oxymatrine group (P = 0.041). In HSC-T6 cells the mRNA and protein expression levels of alpha SMA in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P = 0.013; protein, P = 0.042; respectively); The mRNA and protein expression levels of type I collagen in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P < 0.01; protein, P < 0.01; respectively). CONCLUSION: OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against alpha SMA expression and type I collagen synthesis in HSC-T6 cells than oxymatrine in vitro.     

Combination of small interfering RNA and lamivudine on inhibition of human B virus replication in HepG2.2.15 cells

AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression by combination of siRNA and lamivudine in HepG2.2.15 cells. METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. The transfected cells were cultured in lamivudine-containing medium (0.05 μmol/L) and harvested at 48, 72 and 96 h. The concentration of HBeAg and HBsAg was determined using ELISA. HBV DNA replication was examined by real- time PCR and the level of HBV mRNA was measured by RT-PCR. RESULTS: In HepG2.2.15 cells treated with combination of siRNA and lamivudine, the secretion of HBeAg and HBsAg into the supernatant was found to be inhibited by 91.80% and 82.40% (2.89 ± 0.48 vs 11.73 ± 0.38, P < 0.05; 4.59 ± 0.57 vs 16.25 ± 0.48, P < 0.05) at 96 h, respectively; the number of HBV DNA copies within culture medium was also significantly decreased at 96 h (1.04 ± 0.26 vs 8.35 ± 0.33, P < 0.05). Moreover, mRNA concentration in HepG2.2.15 cells treated with combination of siRNA and lamivudine was obviously lower compared to those treated either with siRNA or lamivudine (19.44 ± 0.17 vs 33.27 ± 0.21 or 79.9 ± 0.13, P < 0.05). CONCLUSION: Combination of siRNA and lamivudine is more effective in inhibiting HBV replication as compared to the single use of siRNA or lamivudine in HepG2.2.15 cells.     

基因重组HBV联合表达反义RNA和显性阴性突变体抗HBV作用及HBV包装细胞系的构建

目的探讨HBV作为基因治疗载体的可能性并检验其联合表达反义RNA和显性阴性突变体抗HBV的作用.方法在表达完整HBV颗粒的质粒上,经基因修饰后联合表达S区反义RNA和核心-p蛋白的融合蛋白,整合于具有HBV复制的2.2.15细胞,形成细胞克隆,ELISA法检测细胞培养上清液中HBsAg和HBeAg,斑点杂交法检测细胞内HBV核壳中HBV DNA,PCR检测上清液中重组HBV颗粒.HBV全基因经删除包装信号ε区后,插入到G418抗性pCI-neo载体,转染HepG2细胞系,用G418筛选形成细胞克隆,检测表达HBsAg及HBcAg较多者作为HBV包装细胞系,进一步转染表达复制缺损型HBV的质粒,经两种抗生素同时筛选,PCR方法观察上清液中的病毒.结果2.2.15-pMEP4组、2.2.15-CP组、2.2.15-SAS组和2.2.15-CPAS组,对HBsAg平均抑制率分别为2.74%±3.83%、40.08%±2.05%(t=35.5,P＜0.01)、66.54%±4.45%(t=42.3,P＜0.01)和73.68%±5.07%(t=51.9,P＜0.01);对HBeAg平均抑制率分别为4.46%±4.25%、52.86%±1.32%(t=36.2,P＜0.01)、26.36%±1.69%(t=22.3,P＜0.01)和59.28%±2.10%(t=39.0,P＜0.01);对HBV复制的抑制率分别为0、82.0%、59.9%和96.6%.在各治疗组培养上清液中均能检测出重组HBV颗粒.证明包装细胞系具有HBsAg和HBcAg表达,pMEP-CPAS质粒转染G418抗性包装细胞系,在细胞培养上清液中检出重组HBV,未检出野生型HBV.结论在同一载体上联合表达S区反义RNA及核心蛋白与部分P蛋白的融合蛋白,具有较单一机制更强的抗HBV作用;经修饰后的HBV基因组在野生型HBV辅助下,仍能包装并分泌完整的HBV样颗粒.包装细胞系能为复制缺损型HBV提供包装,但效率较低.     

减毒水痘病毒对乙型肝炎病毒复制的影响

目的观察减毒水痘病毒对乙型肝炎病毒复制的影响。方法分别用减毒水痘病毒接种鸭乙型肝炎模型和HepG22.2.15细胞,以斑点杂交和EIA方法分别检测鸭血清中DHBVDNA和细胞培养上清液中HBsAg、HBeAg的含量。结果减毒水痘病毒两个剂量组均显示鸭血清病毒量下降,200pfu/kg组在给药后第10天和停药5d时,DHBVDNA吸光度（A）平均值分别由给药前1.17±0.29降至0.59±0.45和0.21±0.21,相比差异有显著性（t＝3.51,P＜0.01和t＝7.54,P＜0.001）;400pfu/kg组在给药后第5、10天DHBVDNA无下降,停药5d时DHBVDNAA平均值由给药前0.70±0.25降至0.32±0.17,差异有显著性（t＝3.58,P＜0.01）;减毒水痘病毒对2.2.15细胞分泌HBeAg、HBsAg均有抑制作用,最大抑制率分别为61%和33%,对HBeAg的抑制较HBsAg强。结论减毒水痘病毒在鸭乙型肝炎模型上可以显著降低血清DHBVDNA水平;在体外能够直接抑制2.2.15细胞分泌HBsAg、HBeAg,提示该病毒可能对乙型肝炎病毒复制有干扰或抑制作用。     

Effective inhibition of expression of hepatitis B virus genes by DNAzymes

AIM: To evaluate the inhibitory effects of DNAzymes on the expressions of hepatitis B virus (HBV) s (HBsAg) and e (HBeAg) in 2.2.15 cells, and to explore the potential therapeutic effects of DNAzymes on replication of HBV genome. METHODS: DNAzymes DrzBS and DrzBC specific to HBV (ayw subtype) s gene ORF A157UG and e gene ORF A1816UG, were designed and synthesized. Inhibitory effects of DrzBS or DrzBC on the expressions of HBV s and e genes as well as HBV DNA levels in culture supernatants were observed in 2.2.15 cells. RESULTS: After being treated with DrzBS or DrzBC, the expression of HBV s or e genes in 2.2.15 cells was depressed dramatically. The maximum inhibition rate was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The concentration for effective inhibition of both DrzBS and DrzBC was within 0.1-2.5 μmol/L, showing a dose-dependence. The efficiency of inhibiting HBsAg, HBeAg in 2.2.15 cells by DrzBS or DrzBC was higher than that of the same target genes by antisense oligonucleotides (ASON). The concentration for effective inhibition of DNAzymes was at least 10-fold lower compared with ASON controls. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed. CONCLUSION: DrzBS and DrzBC can block the expression of HBV s- and e-genes in 2.2.15 cells and provide a specific and effective anti-HBV gene therapeutic means.     

APOBEC3G体外抗乙型肝炎病毒的研究

目的探讨载脂蛋白B mRNA编辑酶催化多肽样蛋白3G（apolipoprotein B mRNA editing enzyme catalytic polypeptide like3G,APOBEC3G）（也称为CEM15）体外抗乙型肝炎病毒（HBV）的作用及其机制。方法脂质体转染pcDNA3.1 Human APOBEC3G-Myc-6Xhis、pcDAN3.1/His-C进入HepG2.2.15细胞,转染后,RT-PCR证实转染基因的表达,Western Blot证实蛋白的表达。通过ELISA方法检测细胞上清液中乙型肝炎表面抗原（HBsAg）及乙型肝炎e抗原（HBeAg）,RT-PCR分析APOBEC3G对HBV mRNA转录的影响。结果APOBEC3G基因与蛋白在HepG2.2.15细胞都有表达,与空质粒转染组相比,pcDNA3.1 Human APOBEC3G-Myc-6Xhis转染组HBsAg含量下降70.38%,HBeAg含量下降62.88%,未转质粒细胞为空白对照组。结论APOBEC3G在体外可以抑制HBV复制,可以作为一种新型的抗病毒制剂治疗乙肝病毒感染。     

Effective inhibition of expression of hepatitis B virus genes by DNAzyrnes

AIM: To evaluate the inhibitory effects of DNAzymes on the expressions of hepatitis B virus (HBV) s (HBsAg) and e (HBeAg) in 2.2.15 cells, and to explore the potential therapeutic effects of DNAzymes on replication of HBV genome. METHODS: DNAzymes DrzBS and DrzBC specific to HBV(aywsubtype) s gene ORF A157UG and e gene ORF A1816UG,were designed and synthesized. Inhibitory effects of DrzBS or DrzBC on the expressions of HBV s and e genes as well as HBV DNA levels in culture supernatants were observed in 2.2.15 cells.RESULTS: After being treated with DrzBS or DrzBC, the expression of HBV s or e genes in 2.2.15 cells was depressed dramatically. The maximum inhibition rate was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The concentration for effective inhibition of both DrzBS and DrzBC was within 0.1-2.5 μmol/L, showing a dosedependence. The efficiency of inhibiting HBsAg, HBeAg in 2.2.15 cells by DrzBS or DrzBC was higher than that of the same target genes by antisense oligonucleotides (ASON). The concentration for effective inhibition ofDNAzymes was at least 10-fold lower compared with ASON controls. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed.CONCLUSION: DrzBS and DrzBC can block the expression of HBV s- and e-genes in 2.2.15 cells and provide a specific and effective anti-HBV gene therapeutic means.