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。     

RNA干扰在动物体内抗乙型肝炎病毒的效果

目的以乙型肝炎病毒(HBV)C区为靶位,动物实验体内观察RNA干扰抗HBV的效果。方法以流体动力学法建立HBV感染的动物模型,将pcDNA 3．1-HBV和体外细胞实验证明有效的小干扰 RNA(siRNA)尾静脉共注射BALB／c小鼠;用时间分辨免疫荧光分析法检测小鼠血清中乙型肝炎表面抗原(HBsAg)水平,用荧光定量聚合酶链反应法检测血清HBV DNA水平,用逆转录聚合酶链反应法检测 HBV C-mRNA,用免疫组织化学法检测肝组织HBsAg和乙型肝炎核心抗原。结果在小鼠体内,siRNA 能有效抑制HBV的复制和表达,干扰效果至少持续3 d。结论靶向HBV C区的siRNA在动物体内能有效抗HBV。     

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。     

针对P基因的siRNA对乙肝病毒S表达稳定性的影响

目的以乙型肝炎病毒（HBV）P基因为靶点，构建表达小干扰RNA（siRNA）的质粒载体psRNA1、psRNA2、psRNA3、psRNA4及对照psiRNA0，体外观察针对P基因的siRNA对HBs表达的影响。方法设计并合成针对HBV-P基因的siRNA寡核苷酸，经退火形成双链后克隆入psiRNA-H1neo质粒中，通过鉴定将构建成功的5个psiRNAs真核表达质粒瞬时转染HepG2．2．15细胞，用半定量RT-PCR对筛选到的位点进行了试验观测抑制效果。结果表明针对P基因的siRNA能下调HBs的表达，其中psiRNA1、psiRNA2的抑制HBs基因表达的效果最强。结论针对P基因的siRNA可以有效的抑制HBs的表达，抑制的效果和siRNA的靶位点有关。     

RNA干扰抑制胰腺癌细胞DNA-PKcs基因表达对放射敏感性的影响

目的探讨稳定表达的siRNA抑制DNA-PKcs基因表达对胰腺癌细胞BxPC-3放射敏感性的影响。方法以人胰腺癌细胞BxPC-3为研究对象,将预先设计的siRNA与质粒连接。构建质粒与阴性对照质粒分别转化大肠杆菌,经克隆、扩增、纯化后转染BxPC-3细胞,潮霉素筛选,以半定量Westernblot检测靶蛋白表达变化,用细胞克隆计数实验检测放射敏感性变化。结果成功构建以DNA-PKcs基因mRNA为靶序列的pSilencer2.1质粒,经潮霉素筛选出稳定的整合目的质粒的BxPC-3细胞,DNA-PKcs蛋白被抑制最大达到83%,实验组D0、Dq及SF2(1.284±0.017、4.006±0.023和0.567±0.045)均明显低于阴性对照组(1.458±0.026、4.840±0.019和0.833±0.076)(P<0.001)。SER为1.47。结论针对DNA-PKcs基因的siRNA表达载体稳定转染人胰腺癌细胞BxPC-3,能够引发DNA-PKcs表达抑制,进而产生放射增敏作用。     

小发夹RNA体外抑制乙型肝炎病毒X蛋白表达的实验研究

目的构建靶向乙型肝炎病毒X基因（HBX）的shRNA表达载体pSilencer3．1-shHBX,观察其体外抑制HBX在HepG2细胞中表达的作用,为应用RNA干扰技术进一步研究HBX基因的功能奠定基础。方法设计并构建靶向HBx的shRNA表达载体pSilencer3．1-shHBX,脂质体转染法将HBX表达载体pcDNA3．1-HBx与pSilencer3．1-shHBX共转染人肝癌HepG2细胞,培养72h后以RT-PCR检测HBX基因表达情况,以Western blot检测HBX蛋白的表达量。结果经酶切和测序鉴定,构建的重组质粒pSilencer3．1-shHBX与设计一致。该质粒使HBX基因mRNA表达量降低47．1％,使HBx蛋白表达量降低58．9％,而阴性对照质粒无此作用。结论成功构建靶向HBXshRNA表达载体pSilencer3．1-shHBX,该质粒可体外抑制HBX在HepG2细胞中的表达。     

靶向cyclinE小干扰RNA对肝癌HepG2细胞增殖和凋亡的影响

目的:以cyclinE基因编码区为靶位,构建表达小干扰RNA(siRNA)的质粒载体,观察转染后对HepG2细胞的影响.方法:针对cyclinE基因序列构建表达siRNA的真核表达载体pSilencer3.1-H1hygro,利用脂质体Metafectene转染CyclinE基因高表达的肝癌细胞株HepG2.流式细胞仪检测细胞周期及凋亡率,MTT法检测细胞增殖活性,RT-PCR和Western blot法观察转染后细胞cyclinE基因表达.结果:成功构建了表达siRNA的真核质粒载体,转染后cyclinE基因mRNA及蛋白表达水平分别下降了79%和65%.结论:靶向cyclinE基因的siRNA可有效沉默HepG2细胞高表达的cyclinE基因,从而抑制肝癌细胞的增殖并促进凋亡.     

小干扰RNA有效抑制乙型肝炎病毒的动物实验

目的 以乙型肝炎病毒（HBV）S区为靶位，观察小干扰RNA（siRNA）在动物体内抗HBV的效果。方法 以流体动力学法建立HBV感染的动物模型，将pcDNA3．1-HBV和细胞体外实验证明有效的siRNA尾静脉共注射Balb／c小鼠，用时间分辨免疫荧光分析法（IFMA）检测小鼠血清中HBsAg，用定量聚合酶链反应法（FQ-PCR）检测血清HBV DNA，用逆转录聚合酶链反应（RT-PCR）法检测HBV S-mRNA，用免疫组织化学法检测肝组织HBsAg和HBcAg。结果 在小鼠体内，siRNA能有效抑制HBsAg的分泌，降低HBVDNA的滴度，免疫组织化学结果也证实HBsAg、HBcAg刚性细胞数明显减少，干扰效果至少持续3d，而无关siRNA则无抑制作用。结论 在动物体内靶向HBV S区的siRNA能有效特异抑制HBV。     

RNA干扰抑制胰腺癌细胞DNA-PKcs基因表达对放射敏感性的影响

目的探讨稳定表达的siRNA抑制DNA-PKcs基因表达对胰腺癌细胞BxPC-3放射敏感性的影响.方法以人胰腺癌细胞BxPC-3为研究对象,将预先设计的siRNA与质粒连接.构建质粒与阴性对照质粒分别转化大肠杆菌,经克隆、扩增、纯化后转染BxPC-3细胞,潮霉素筛选,以半定量Western blot检测靶蛋白表达变化,用细胞克隆计数实验检测放射敏感性变化.结果成功构建以DNA-PKcs基因mRNA为靶序列的pSilencer2.1质粒,经潮霉素筛选出稳定的整合目的质粒的BxPC-3细胞,DNA-PKcs蛋白被抑制最大达到83%,实验组D0、Dq及SF2(1.284 ± 0.017、4.006 ± 0.023和0.567 ± 0.045)均明显低于阴性对照组(1.458 ± 0.026、4.840 ± 0.019和0.833 ± 0.076)(P ＜ 0.001).SER为1.47.结论针对DNA-PKcs基因的siRNA表达载体稳定转染人胰腺癌细胞BxPC-3,能够引发DNA-PKcs表达抑制,进而产生放射增敏作用.     

针对HBV S基因发夹状siRNAs表达载体的构建及其在HepG2215细胞中对HBV基因表达的抑制作用

目的探讨通过载体在体外培养的细胞中表达发夹状siRNAs对HBV基因表达的抑制作用。方法构建表达针对HBV S基因mRNA的发夹状siRNAs的质粒pSilencer 2.1-U6-S,并与ayw亚型HBV全基因组表达质粒共转染体外培养的HepG2215细胞,用半定量RT-PCR分析目标mRNA表达丰度的变化,用ELISA法观察HBsAg表达的变化。结果siRNA处理组细胞上清中HbsAg和HBeAg含量分别比空白对照组降低了63.4%和68.0%,细胞中的2.1kb的信使RNA降低了75.2%。结论siRNA在体外培养的细胞中能有效地抑制HBV基因的表达。     

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。     

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery

RNA interference is highly effective at inhibiting HBV gene expression and replication. However, before small interfering RNA (siRNA) can be used in the clinic, it is essential to develop a system to target their delivery. Antibody-mediated delivery is a novel approach for targeting siRNA to appropriate cells. In this report, we asked whether this siRNA delivery strategy would be effective against HBV. Of 5 candidates, a specific siRNA that effectively inhibited HBV gene expression and replication was determined. Two fusion proteins, s-tP and sCkappa-tP, were constructed to contain a single chain of the human variable fragment, scFv, against hepatitis B surface antigen (HBsAg), a truncated protamine (tP), and in the case of sCkappa-tP, a constant region of the kappa chain (Ckappa). S-tP and sCkappa-tP were developed to provide targeted delivery of the siRNA, siRNA expressing cassettes (SEC), and siRNA-producing plasmids. Fluorescein isothiocyanate-siRNA, fluorescein isothiocyanate-SEC, and plasmid DNA were specifically delivered into HBsAg-positive cells using the sCkappa-tP fusion protein, and effectively inhibited HBV gene expression and replication. HBV gene expression was also inhibited by siRNA or siRNA-producing plasmids in HBV transgenic mice. CONCLUSION: Our results describe a potential method for the targeted delivery of siRNA or siRNA-producing plasmids against HBV, using anti-HBsAg fusion proteins.     

Effect of vector-expressed siRNA on HBV replication in hepatoblastoma cells

AIM: To study the effect of siRNA expressed from DNA vector on HBV replication. METHODS: Human U6 promoter was amplified from genomic DNA and cloned into plasmid pUC18 to construct a mammalian siRNA expression vector pUC18U6. Then oligonucleotides coding for a short hairpin RNA against HBV were cloned into pUC18U6 to form pUC18U6HBVsir which was introduced into 2.2.15 cells by using liposome-mediated transfection. 2.2.15 cells transfected by pUC18U6 and pUC18U6GFPsir which expressed siRNA against green fluorescent protein and mock-transfected 2.2.15 cells were used as controls. Concentration of HBsAg in the supernatant of the transfected cells was measured by using solid-phase radioimmunoassay. RESULTS: A mammalian siRNA expression vector pUC18U6 was constructed successfully. Compared with controls, pUC18U6HBVsir which expressed siRNA against HBV decreased concentration of HBsAg significantly by 44%(P<0.05). CONCLUSION: HBV replication in 2.2.15 cells is inhibited by siRNA expressed from the DNA vector.     

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.