细胞内La蛋白对乙型肝炎病毒蛋白质表达的影响

目的 探讨细胞内La蛋白对HBV蛋白质表达的影响.方法 针对人La蛋白序列设计特异性的小分子干扰RNA(siRNA),通过体外转录的方法合成双链siRNA,并转染进入稳定表达HBV的HepG2.2.15细胞,实时荧光定量基因扩增技术测定HepG2.2.15细胞中的La蛋白mRNA变化水平;电化学发光分析技术检测HepG2.2.15细胞分泌的HBsAg和HBeAg变化情况.所得数据进行配对假设检验的统计学分析.结果 特异性siRNA干扰La蛋白mRNA的表达,使La蛋白在HepG2.2.15细胞中的表达降低;HepG2.2.15细胞分泌在培养上清液中的HBsAg和HBeAg表达显著下降,相关性分析显示,HBsAg、HBeAg变化水平与La蛋白mRNA变化水平呈正相关,相关系数分别为0.938和0.899.结论 特异性siRNA能够抑制细胞内La蛋白mRNA的表达,La蛋白可通过某种方式影响HBV蛋白质的表达.     

甲胎蛋白启动子介导反义乙型肝炎病毒X基因在肝癌细胞中的表达及其作用

目的 构建含甲胎蛋白(AFP)启动子和增强子的反义乙型肝炎病毒X基因(HBX)真核表达载体，研究其特异性和有效性，为开发肝癌细胞特异性HBX反义RNA基因治疗乙型肝炎病毒(HBV)奠定基础。方法 聚合酶链反应(PCR)扩增HBX(1370—1872nt)基因，克隆至EB病毒表达载体，双轮PCR筛选、鉴定基因插入方向。脂质体转染肝癌细胞和ECV304细胞，Northernblot检测HBX mRNA的表达，酶联免疫试验(ELISA)检测HBV抗原，荧光定量PCR检测HBV DNA。结果 成功构建正、反义RNA表达载体pEBAF—s—HBX、pEBAF—as—HBX。Northernblot证实反义RNA仅在AFP阳性的肝癌细胞中表达。pEBAF—as—HBX转染3d后，可显著抑制2．2．15细胞HBV复制和抗原表达，其HBsAg、HBeAg抗原表达较正义对照分别下降37．9％和36．8％，HBV DNA降低25％。结论 反义RNA表达载体pEBAF—as—HBX仅在肝癌细胞中特异表达、并可有效抑制HBV，有良好的开发应用前景。     

Inhibition of hepatitis B virus expression and replication by RNA interference in HepG2.2.15

INTRODUCTION Hepatitis B is a severe infectious disease threatening peoples’ health all over the world. There is still no efficient therapy to control HBV persistent replication, which may lead to the development of liver cirrhosis and hepatocellualar ca…     

针对HBV X基因的siRNA抑制HepG2细胞HBV的复制和表达

目的构建能转录产生针对乙型肝炎病毒（HBV）X基因转录体的小干扰RNA（siRNA）转录载体pSIH—BV／X,研究其在体外对HBV复制和抗原表达的抑制作用。方法将构建成功的pSIHBV／X与HBV1．3倍体真核表达质粒pHBV1．3共转染HepG2细胞，转染后24、48、72h检测上清液中HBsAg、HBeAg的变化，并检测72h时HBV DNA的变化。结果成功构建了针对HBVX基因转录体的siRNA表达载体pSIHBV／X，并发现它能抑制HBsAg、HBeAg的分泌，抑制高峰在72h．抑制率分别是64％和61％，而无关对照序列的siRNA无此作用。荧光定量PCR证实HBV DNA的复制亦受到抑制，抑制率31％。结论针对HBVX基因转录体的siRNA在体外具有抑制HBV复制和抗原表达的作用。     

Inhibition of hepatitis B virus replication in 2.2.15 cells by expressed shRNA

Summary.  Hepatitis B virus (HBV) infection is a worldwide health problem. To determine whether RNA interference (RNAi) could inhibit ongoing HBV replication in 2.2.15 cells, we constructed shRNA-producing vector pU6P based on the mouse U6 RNA promoter and cloned 12 targeted sequences against HBV into the vector, resulting in a series of pU6-siHBV vectors. The recombinant vectors were transfected into 2.2.15 cells, HBsAg and HBeAg in cultured media were assayed using enzyme-linked immunosorbent assay at various days after transfection. The amount of HBV DNA in the culture medium was quantitated by real-time polymerase chain reaction. HBsAg and HBeAg expression were inhibited by 72.8 ± 5.4% ( P  = 0.00003) and 55.8 ± 6.2% ( P  = 0.000026), respectively, 4 days after transfection with pU6-siHBV5. The greatest inhibition of HBV DNA was decreased by approximately 1.9-fold ( P  = 0.013) on day 6 post transfection with pU6-siHBV11 compared with that of empty vector. No change was found for HBV protein expression and DNA replication on pU6-siGFP (negative control) transfected cells. Our data demonstrate that the transfection of HBV-targeted shRNA-producing vector in 2.2.15 cells could inhibit the HBV protein expression and HBV DNA replication specifically. RNAi may be considered as a potential antiviral approach for human HBV infection.     

Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro

AIM： To explore the inhibitory effects of pokeweed antiviral protein seed （PAP-S） and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus （HBV） replication in vitro. METHODS： HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold overlength plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS： The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 μg/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 μg and 2.0 μg plasmid pXF3H-PAP, the levels of HBV nucleocapside- associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION： Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.     

RNA干扰技术用于抗乙型肝炎病毒的实验研究

目的 构建针对乙型肝炎病毒表面抗原(HBsAg)和核心抗原的小干扰RNA(siRNA)表达载体pSuper-C，观察其对HepG2 2．2．15细胞(简称2、2．15细胞)中HBV DNA转录和翻译相应蛋白的影响。方法 根据RNA干扰(RNAi)作用原理设计针对HBV核心区的相应序列，再将其克隆入含聚合酶ⅢH1-RNA启动子的真核表达载体pSuper，将此重组质粒以电转染法转入2．2．15细胞中，用酶联免疫吸附法(Abbott试剂)检测培养上清液中HBsAg和e抗原(HBeAg)的表达。结果 经酶切鉴定、电泳和测序分析证明，成功构建了含作用序列的重组质粒pSuper-C；但以电穿孔法转染 2．2．15细胞后末能发现其对2．2．15细胞培养上清液中的HBsAg和HBeAg的表达有影响。结论 RNAi在2．2．15细胞中的作用还需进一步的实验来证实。     

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.     

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.     

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。     

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.     

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.     

乙型肝炎病毒(HBV)反基因与HBV的结合及对HBV复制的影响

目的 研究三螺旋形成寡核苷酸与HBV基因的结合情况及其对HBV复制的影响。方法 全盛一段能与HBV核心启动子位点（1734-1753nt)形成三螺旋的寡核苷酸（TF020）并标记上生物素分别用脂质体-TF020及裸TF020转染HepG2.2.15细胞,用链酶亲和素-生物素法（SABC）检测其转染效率及其与HepG2.2.15细胞中HBVDNA的结合情况;并采用ELISA、半定量逆转录（RT）-PCR及荧光定量PCR法分别检测经寡核苷酸处理的HepG2.2.15细胞及空白对照细胞上清HBsAg、HBeAg、HBV DNA及细胞中HBV RNA的水平。结果 脂质体-TF020转染HepG2.2.15细胞的效率可达80%,而裸TFO20转染率最高为40%;TF020主要定位于细胞核及胞浆中,TF020处理组细胞上清中HBsAg、HBeAg含量分别较空白对照组降低37.0%及78.2%,HBV DNA水平较空白对照组明显降低;而细胞中2.4kb/2.1kb RNA、3.5kb/3.4kbRNA亦分别降低31.6%及70.2Z%。结论 TF020通过脂质体包裹后可以很好地进入细胞并与HBV DNA结合;TF020能够有效地抑制HBV复制,发挥抗病毒作用。     

Knockdown of HBV surface antigen gene expression by a lentiviral microRNA‐based system inhibits HBV replication and HCC growth

Summary. Current options for the treatment of hepatitis B virus (HBV) infections, a common liver cancer risk factor, are limited. While RNA interference (RNAi) technologies have been shown to inhibit HBV replication, the consequent effects on hepatocellular carcinoma (HCC) cell growth are not fully understood. The aim of this study was to evaluate the effect of RNAi‐mediated decrease in the HBV surface antigen (HBsAg) gene on HBV replication and HCC growth. A lentiviral microRNA‐based system expressing siRNAs targeting the HBsAg gene (LVshHBS) was developed and transfected into HepG2.2.15 cells (HBV stably expressing line). We found that LVshHBS significantly inhibited the HBsAg mRNA and protein levels in the HepG2.2.15 cells, while HBsAg secretion into the culture supernatant decreased by 70%. BALB/c (nu/nu) mice were injected with HepG2.2.15 cells transduced with LVshHBS or control vectors to investigate the effect of inhibiting the HBsAg on the development of tumour growth in a human HCC nude mice model. Compared with the control, the tumour growth in nude mice was significantly decreased after injection with LVshHBS. Microarray analysis of tumour‐related genes in LVshHBS‐transduced HepG2.2.15 cells showed that the expressions of genes involved in cell cycle, differentiation and oncogenesis such as ACP2, BHLHB2, CLK3, CTSC, FOS, NR1D1, PIM1 and SEPT6 genes were downregulated, while that of the E2F3 gene was upregulated. In conclusion, lentiviral microRNA‐based RNAi against the HBsAg gene not only inhibits HBV replication but also inhibits the growth of HCC. Downregulation of growth‐related genes is implicated in this mechanism of inhibition.     

针对HBV—P基因的RNA干扰抑制乙型肝炎病毒的研究

目的构建针对乙型肝炎病毒P基因编码区、能在体内转录产生发夹状小干扰RNA（siRNA）的表达载体psiHBV／p,观察RNA干扰对HBsAg、HBeAg及乙型肝炎病毒DNA复制的抑制作用。方法针对HBV—P基因区特异序列,构建siRNA的表达载体psiHBV／p。采用脂质体介导方法将其与1．3倍HBV真核表达质粒pHBV1．3共转染HepG2细胞。分别于转染后24小时、48小时、72小时用ELISA法对HepG2细胞培养上清液进行HBsAg、HBeAg的检测;于转染后72小时通过FQ-PCR法分析RNA干扰作用对HBVDNA的抑制效果。结果成功构建了针对HBV—P基因区的siRNA的真核表达重组体psiHBV／p,并发现它能明显抑制HBsAg及HBeAg的分泌,转染后第二天抑制率达高峰,分别为84％、65％。FQ—PCR结果也证实了转染72小时后,随psiHBV／p比例的升高,其对HBV DNA的抑制作用也随之增加。结论成功构建的psiHBV／p,它能在体内持续转录产生针对P基因转录体的发夹状siRNA;在细胞水平上,体内转录产生的、针对乙型肝炎病毒P基因区特异序列的siRNA对共转染的重组载体pHBV1．3有显著和特异的抑制作用。     

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.     

短发夹状双链RNA抑制丙型肝炎病毒IRES介导的基因表达

目的研究载体表达的短发夹状双链RNA（shRNA）对丙型肝炎病毒（HCV）IRES介导的基因表达的特异性抑制作用。方法构建HCV IRES调控的绿色荧光蛋白表达载体（pIRES—GFP）和虫荧光索酶表达载体（p5′ UTR—Luc），以及针对HCV IRES的shRNA表达载体（pshRNA-HCV）。共转染HepG2细胞，于转染后24、48、72h观察绿色荧光的强弱，用Western blot检测绿色荧光蛋白的表达，半定量逆转录聚合酶链反应法检测GFP的mRNA水平。双荧光索酶系统检测虫荧光索酶活性。结果pshRNA-HCV作用组绿色荧光强度明显弱于未干扰组，GFP蛋白表达量及虫荧光素酶活性降低60％～70％，半定量逆转录聚合酶链反应显示pshRNA-HCV导致了GFP基因mRNA水平的降低。结论针对HCV IRES的shRNA能够显著和特异地抑制该区域调控的蛋白表达水平及mRNA水平，该研究结果为利用RNA干扰技术治疗HCV感染进行了初步探索。