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。     

载脂蛋白质B mRNA编辑酶催化多肽3G抑制乙型肝炎病毒和鸭乙型肝炎病毒复制

目的了解载脂蛋白质BmRNA编辑酶催化多肽3G（APOBEC3G）对乙型肝炎病毒（HBV）和鸭乙型肝炎炎病毒（DHBV）复制的抑制作用。方法从健康人外周血单个核细胞提取RNA，逆转录聚合酶链反应扩增APOBEC3G，将产物克隆到pXF3H载体的EcoRⅠ和Hind Ⅲ酶切位点以构建真核表达质粒；以ayw亚型HBV全长质粒构建具有复制能力的1．3倍HBV质粒（pHBV1．3）。不同剂量的APOBEC3G真核表达质粒与pHBV1．3共转染HepG2细胞；酶联免疫吸附法检测细胞培养上清液的乙型肝炎表面抗原和e抗原水平，Southernblot和Northernblot分析HBV核衣壳相关DNA和RNA的水平变化。不同剂量APOBEC3G真核表达质粒与头尾相接的2倍DHBV质粒共转染LMH鸡肝癌细胞，Southernblot分析DHBV核衣壳相关DNA水平变化。结果成功构建APOBEC3G真核表达质粒和具有复制能力的1．3倍HBV质粒。APOBEC3G抑制乙型肝炎表面抗原和e抗原的分泌，转染细胞内HBV核衣壳相关RNA表达水平下降，而对核心蛋白质的表达没有影响；APOBEC3G对转染细胞内HBV和DHBV核衣壳相关DNA水平具有剂量依赖的抑制效应。结论APOBEC3G对HBV和DHBV复制具有抑制作用。     

肽核酸抑制HBV复制与表达的体外研究

合成肽核酸(PNA)片段,由OligofectamineTm转染至HepG2.2.15细胞与HBV各编码区保守区域相结合。用ELISA方法测定HepG2.2.15细胞中HBsAg、HBeAg滴度,PCR测定HBV DNA含量。通过计算抑制百分率,筛选有效PNA片段。结果在500 mol/L浓度下,PG3 HBsAg、HBeAg、HBV DNA表达抑制率分别为51.03%、55.38%、20.10%;PG6 HBsAg、HBeAg、HBV DNA表达抑制率分别为47.00%、43.10%、51.03%。表明PNA可由OligofectamineTm转染至HepG2.2.15细胞,从而抑制HBV的复制和抗原系统的表达。     

青蒿琥酯对乙型肝炎病毒复制及肝癌细胞株HepG2.2.15凋亡的影响

目的 探讨青蒿琥酯在体外对乙型肝炎病毒复制及肝癌细胞株HepG2.2.15凋亡的影响.方法 将不同浓度青蒿琥酯作用于转染乙型肝炎病毒全基因组DNA的肝癌细胞株HepG2.2.15,收集48 h上清,采用酶联免疫吸附实验检测上清中乙型肝炎病毒表面抗原(HBsAg)和e抗原(HBeAg),采用荧光定量PCR法检测HBV-DNA,流式细胞术检测细胞凋亡.结果 青蒿琥酯对HBV复制具有抑制作用,随着浓度增加,对HBsAg和HBeAg的抑制率逐渐上升,细胞内HBV-DNA 复制水平下降;青蒿琥酯可诱导肝癌细胞早期凋亡及导致细胞死亡,随浓度增加,HepG2.2.15细胞早期凋亡率及死亡率均增加.结论 青蒿琥酯对HepG2.2.15细胞HBsAg和HBeAg的分泌及HBV-DNA复制具有抑制作用,并具有诱导HepG2.2.15细胞凋亡的作用.     

胶原水凝胶在HepG2.2.15细胞三维培养中的应用研究北大核心CSCD

目的了解用胶原水凝胶为支架构建HBV转染细胞HepG2.2.15的三维(three-dimensiona,3D)培养模型的应用情况。方法采用扫描电镜(SEM)和原子力显微镜(AFM)观察水凝胶内胶原纤维的结构特征,以胶原水凝胶为支架建立HepG2.2.15细胞3D培养模型。采用倒置显微镜观察细胞形态特征;采用MTS法和钙黄绿素-AM(Ca-AM)染色法检测细胞的增殖活性;采用ELISA法检测细胞HBsAg、HBeAg的分泌;采用荧光探针PCR法检测HBV DNA的表达。结果HepG2.2.15细胞在96孔板胶原水凝胶中能培养45d,在24孔板内培养则长达160d,且生长良好,并能维持转染细胞特性,持续分泌HBsAg、HBeAg和HBV DNA。结论建立的胶原水凝胶HepG2.2.15细胞3D培养模型能长时间维持细胞增殖。     

胶原水凝胶在HepG2.2.15细胞三维培养中的应用研究

目的了解用胶原水凝胶为支架构建HBV转染细胞HepG2.2.15的三维(three-dimensiona, 3D)培养模型的应用情况。方法采用扫描电镜(SEM)和原子力显微镜(AFM)观察水凝胶内胶原纤维的结构特征,以胶原水凝胶为支架建立HepG2.2.15细胞3D培养模型。采用倒置显微镜观察细胞形态特征;采用MTS法和钙黄绿素-AM(Ca-AM)染色法检测细胞的增殖活性;采用ELISA法检测细胞HBsAg、HBeAg的分泌;采用荧光探针PCR法检测HBV DNA的表达。结果 HepG2.2.15细胞在96孔板胶原水凝胶中能培养45 d,在24孔板内培养则长达160 d,且生长良好,并能维持转染细胞特性,持续分泌HBsAg、HBeAg和HBV DNA。结论建立的胶原水凝胶HepG2.2.15细胞3D培养模型能长时间维持细胞增殖。     

地塞米松、环磷酰胺和他克莫司三种免疫抑制剂影响HBV复制的体外研究

目的探讨地塞米松(DEX)、环磷酰胺(CYP)和他克莫司(FK506)在体外对HBV蛋白合成、DNA复制的影响。方法将不同浓度的DEX、CYP和FK506作用于HepG2.2.15细胞系,通过MTT实验确定药物安全浓度范围,通过检测细胞培养上清液中HBsAg和HBeAg水平,以及细胞内HBV DNA水平来评价HBV的表达和复制情况。结果DEX、CYP和FK506的药物安全浓度范围分别为0~500、0~1000和0~10μg/mL。与培养基对照处理相比,FK506处理后HBsAg、HBeAg分泌及HBV DNA复制变化差异无统计学意义;CYP处理增加HBsAg分泌,与对照相比差异有统计学意义(P0.05);但HBeAg分泌及HBV DNA复制变化差异无统计学意义;DEX作用减少HBsAg和HBeAg的分泌,与对照相比差异有统计学意义(P0.05);但是细胞内HBV DNA复制水平差异无统计学意义(P0.05)。结论在药物安全浓度范围内,FK506对HepG2.2.15细胞HBV DNA复制无直接抑制或促进作用。CYP处理后增加HepG2.2.15细胞HBsAg表达,但HBeAg表达及HBV DNA复制变化差异无促进作用;DEX处理抑制HepG2.2.15细胞HBsAg和HBeAg表达,但对HBV DNA复制无抑制作用。     

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.     

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。     

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.     

Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo

AIM: To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model. METHODS: The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. RESULTS: There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with In vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log 10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups. CONCLUSION: These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.     

N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

INTRODUCTION Hepatitis B virus (HBV) infects more than 350 million people worldwide and is a leading cause of end-stage liver disease and of hepatocellular carcinoma[1]. HBV is non- cytopathic for hepatocytes; however, most newly HBV-infected adult patien…     

靶向S区和C区基因的M1GS RNA核酶共同抑制HBV的表达

目的:研究靶向HBVS区和C区基因的M1GSRNA核酶共同作用对HBV基因表达的影响.方法:选择HBVayw亚型S区基因294nt和C区基因2333nt为切割位点,以含有编码M1RNA的DNA序列的质粒pTK117为模板,通过PCR扩增得到M1GSRNA核酶的DNA模板,并将其克隆至真核表达载体pEGFP-C1得到重组质粒pEGFP-GSS和pEGFP-GSC.将2个重组质粒共转染HepG2.2.15细胞,转染后ELISA法测细胞培养液中的HBsAg和HBeAg,RT-PCR检测HBVmRNA.结果:成功构建了分别靶向HBVS区基因和C区基因的真核表达载体.共转染HepG2.2.15细胞后,HBsAg和HBeAg的表达分别被抑制了33.2%和39.1%,HBVCmRNA和SmRNA分别被抑制了32.5%和29.7%,而HepG2.2.15细胞的增殖无明显变化.结论:靶向HBVS区和C区基因的M1GSRNA核酶共同作用可特异性抑制HBVS区和C区基因的表达.     

长片段RNA抑制HepG2.2.15细胞中HBV基因的表达和复制

目的 评估长的反义RNA干扰片段在培养细胞株中对HBV复制的抑制效应.方法将HBV基因组S区的全部核苷酸序列插入至pTARGETTM载体中,并将重组载体转染入HepG2.2.15细胞中.用酶联免疫吸附法检测HBsAg与HBeAg水平,用荧光定量PCR法检测HBVDNA水平.对数据采用多个独立样本Kruskal-Wallis检验与两两比较的Mann-Whitney U检验.结果 经过处理后,HepG2.2.15细胞上清液中HBsAg表达量(A值)在HBS2组(携带长片段反义RNA)为0.621±0.027,在HBS4组(携带正义RNA)为3.399±0.018,对照组为2.232±0.187;HBeAg表达量(A值)在HBS2组、HBS4组和对照组分别为0.749±0.019、1.548±0.025和1.570±0.044; HBV DNA水平(×104拷贝/ml)在HBS2组、HBS4组、对照组分别为1.597±0.082、3.381±0.297和3.610±0.063.与对照组相比,HBS2组HBsAg、HBeAg和HBV DNA表达量均降低,统计量Z值均为-2.309,P值均＜0.05; HBS4组HBsAg表达量增高(Z=-2.309,P＜0.05),而HBeAg和HBV DNA表达量无明显差异,统计量Z值分别为-0.866、-1.155,P值均＞0.05.结论 长片段反义RNA能抑制HBV基因的表达和病毒复制.

Abstract：

Objective To evaluate the inhibitory effects of long antisense RNA on HBV replication in HepG2.2.15 cells. Methods The coding region of HBV S gene was cloned into pTARGET vector in sense and antisense orientations and the recombinant plasmids were transfected into HepG2.2.15 cells which were divided into HBS2 (antisense RNA) group, HBS4 (sense RNA) group and control group. HBsAg and HBeAg in the culture supernant were detected by ELISA. The HBV DNA in the supernant was quantified by real-time PCR. Results After treatment, the levels of HBsAg in HepG2.2.15 cell supernatants of three groups were 0.621 ± 0.027, 3.399 ± 0.018 and 2.232 ± 0.187 respectively; the levels of HBeAg were 0.749 ± 0.019,1.548 ± 0.025 and 1.570 ± 0.044 respectively and the levels of HBV DNA were 1.597 ± 0.082, 3.381 ± 0.297 and 3.610 ± 0.063 respectively. The expressions of HBsAg and HBeAg and the HBV DNA level in HBS2 group were remarkably reduced as compared to the control (Z = -2.309, P ＜ 0.05); whereas the sense plasmid transfection (HBS4) did not affect HBeAg (Z= -0.866) and HBV DNA (Z = -1.155) levels in the culture supernant but slightly increased the HBsAg level (Z = -2.309). Conclusion Antisense RNA might be a useful tool to repress HBV replication.     

Inhibition of hepatitis B virus gene expression and replication by artificial microRNA

AIM： To investigate the inhibitory effects of hepatitis B virus （HBV） replication and expression by transfecting artificial microRNA （amiRNA） into HepG2.2.15 cells. METHODS： Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2.15 cells. HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays （TRFIA）. HBV DNA replication was examined by ？ uorescence quantitative PCR, and the level of HBV S mRNA was measured by semi- quantitative RT-PCR. RESULTS： The efficiency of transient transfection of the vectors into 2.2.15 cells was 55%-60%. All the vectors had significant inhibition effects on HBsAg and HBeAg at 72 h and 96 h after transfection （P 〈 0.01 for all）. The secretion of HBsAg and HBeAg into the supernatant was inhibited by 49.8% ± 4.7% and 39.9% ± 6.7%, respectively, at 72 h in amiRNA- HBV-S608 plasmid transfection group. The copy of HBV DNA within culture supernatant was also significantly decreased at 72 h and 96 h after transfection （P 〈0.01 for all）. In the cells with stable transfection, the secretion of HBsAg and HBeAg into the supernatant was significantly inhibited in all three transfection groups （P 〈 0.01 for all, vs negative control）. The copies of HBV DNA were inhibited by 33.4% ± 3.0%, 60.8% ± 2.3% and 70.1% ± 3.3%, respectively. CONCLUSION： In HepG2.2.15 cells, HBV replication and expression could be inhibited by artif icial microRNA targeting the HBV S coding region. Vector-based artificial microRNA could be a promising therapeutic approach for chronic HBV infection.     

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.     

HDV核酶对乙型肝炎病毒抑制作用的研究

目的研究HDV核酶在细胞内对乙型肝炎病毒(HBV)复制及其抗原表达的抑制作用。方法1)以HBV前基因组mRNA为靶基因,体外筛选出HDV核酶有效作用位点,构建HDV核酶并进行体外测活;2)分别选用tRNA-Val、U6和hCMV 3种真核启动子,重组构建HDV核酶真核表达载体ptVHRz、pSURz和pcDHRz,分别用3种载体转染HepG2.2.15细胞;3)用点杂交、ELISA和实时荧光定量PCR方法分别检测核酶在细胞内的表达及对HBV的抑制作用。结果在HBV C基因区筛选到一位点,所构建的HDV核酶在体外条件下对该位点能产生有效切割。3种核酶表达载体在细胞内均能高效表达,在转染48 h后,ptVHRz和pcDHRz对HBeAg的表达产生了明显的抑制作用,而对HBsAg没有抑制作用。三者对HBV的复制均未产生明显影响。结论HDV核酶在细胞内对HBV抗原的表达能产生特异性抑制作用,但未能有效抑制HBV的复制,对其原因需进一步深入研究。