联合应用siRNA和拉米夫定抑制HBV复制和表达的实验研究

观察联合应用小干扰RNA和拉米夫定对HepG2.2.15细胞中HBV抗原表达和复制的抑制作用。构建并转染重组质粒psil-HBV到HepG2.2.15细胞中。转染后的细胞培养基中加入拉米夫定(0.05μm),分别于48、72、96 h收获细胞。用ELISA方法检测HBeAg和HBsAg;HBV DNA水平用实时定量PCR测定;用逆转录PCR检测HBV mRNA水平。96 h后联合应用小干扰RNA和拉米夫定组细胞培养上清中HBeAg和HBsAg抑制率分别为91.8%和82.4%(P<0.05);HBV mRNA表达水平明显降低。HepG2.2.15细胞中联合应用小干扰RNA和拉米夫定对HBV复制的抑制作用比单独应用siRNA或拉米夫定更有效。     

反义前S/S基因转移表达抗乙型肝炎病毒的研究

为了观察反义基因转录表达抗乙型肝炎病毒(HBV)的作用,将HBV ayw前S/S基因(preS/S)片段反向插入逆转录病毒载体质粒,构建preS/S反义基因重组体,经转染PA317细胞后,获得重组逆转录病毒颗粒.用重组病毒转导2.2.15细胞后,第3天即可见细胞培养上清HBV表面抗原(HBsAg)和e抗原(HBeAg)表达量减少,到转导后第5天,细胞培养上清中的HBsAg和HBeAg表达量降到最低水平,HBsAg抑制率为71%,HBeAg抑制率为27%,抑制作用至少可持续到转导后第11天.空载及正向插入基因的重组逆转录病毒转导对2.2.15细胞内HBV抗原表达无抑制作用.     

特异性表达siRNA的新型乙肝核酸药物的研究

旨在研究靶向于HBsAg的肝脏特异性siRNA,实现核酸药物高靶向性、高特异性基因治疗乙肝。通过重组PCR的方法构建靶向于HBsAg的肝脏特异性表达的siRNA的核酸药物。将目标载体转染HepG2 2.2.15细胞,用ELISA的方法测定转染细胞培养液内HBsAg的含量。PCR鉴定和测序确定重组PAAV-MCS载体PApoE1-ApoE2-hAAT-siHBsAg-U1 3’Box构建成功。重组质粒对HepG2 2.2.15细胞分泌的HBsAg所产生的抑制作用自转染后5 d达到高峰,抑制率达到(30±0.28)%(P<0.01),siRNA特异性表达单元能显著降低HepG2 2.2.15细胞HBV DNA的拷贝数。成功构建的肝脏特异性、siRNA高表达的核酸药物可以有效地抑制HBV基因的表达和复制,为乙肝的进一步治疗研究做了关键性工作。     

人工microRNA抗乙型肝炎病毒效果初探

目的:设计靶向乙型肝炎病毒(HBV)基因保守区的人工microRNA(amiRNA),考察其对HBV基因表达的抑制作用。方法:比对HBV全基因组现有序列,选择保守区设计amiRNA,定向克隆到pcDNA6.2-GW/EmGFP-miR载体,将amiRNA载体与HBV复制载体pHBV1.31共转染HepG2细胞,72 h后收取细胞上清,ELISA检测HBV表面抗原(HBsAg)及e抗原(HBeAg)的含量,荧光定量PCR检测HBV DNA含量。结果:amiRNA可显著抑制细胞上清HBsAg、HBeAg和HBV DNA的水平。结论:amiRNA作为防治HBV感染的潜在有效手段之一值得进一步深入研究。     

利用pSOS系统筛选靶向HBV病毒X基因的siRNA

构建靶向乙肝病毒(HBV)X基因的真核表达载体pSOS-X-siRNA和pSOS-siRNA,转染肝癌细胞系HepG2和HepG2.2.15,筛选和验证高效siRNA。设计4个靶向X基因siRNA,将siRNA和HBx基因插入载体pSOS得重组质粒pSOS-X-siRNA;pSOS-X-siRNA经PacI酶切去除目的片段HBx后得pSOS-siRNA。将质粒pSOS-X-siRNA和pSOS-siRNA分别转HepG2和HepG2.2.15肝癌细胞株。荧光显微镜下观察HepG2细胞绿色荧光(GFP)减弱程度预估干扰效率。ELISA检测HepG2.2.15细胞上清HBsAg、HBeAg表达,Western blotting检测胞内蛋白HBsAg、HBcAg表达,Real time PCR检测胞内HBsmRNA、HBx mRNA的转录。转染4d后,siRNA4使HepG2细胞的GFP信号表达程度最低,为阴性对照的9%,预测其干扰效果最强。siRNA4使HepG2.2.15细胞转染后4d上清的HBsAg蛋白表达为对照的(13.92±1.14)%(P0.05)、HBeAg为(21.69±4.92)%(P0.05),胞内的HBsAg、HBcAg蛋白表达量灰度比值为0.175±0.025、0.0825±0.028,均为各处理组中最低(P0.01),HBs mRNA和HBx mRNA分别降为对照的0.237±0.028(P0.01)、0.110±0.022(P0.01),差异有统计学意义,证实siRNA4为高效干扰位点。利用pSOS成功筛选并验证构建靶向HBV X基因的siRNA靶点。     

靶向ASGPR的反义核酸与抗HBV药物联合用药的体外抗HBV作用

研究以宿主基因ASGPR为靶的反义核酸与抗乙肝病毒(hepatitis B virus,HBV)药物联合应用的抗-HBV作用,为HBV感染的联合治疗和用药提供新的思路。以HepG2.2.15细胞为靶细胞,脂质体为载体将靶向ASGPR的硫代反义寡核苷酸(ASODN)转染至HepG2.2.15细胞中,6h后加入抗病毒药拉米夫定(3TC)或阿地福韦(ADV),72h后收集细胞培养液,采用酶联免疫法及荧光定量PCR法测定ASODN与抗-HBV药物联合应用后对细胞培养液中HBsAg、HBeAg及细胞分泌HBV DNA的抑制作用。采用金正均Q值法对数据进行分析。拉米夫定(3TC)和阿地福韦(ADV)分别与ASODN联用对HBsAg的抑制呈相加或协同作用。随着ADV浓度的增加,两药的协同作用下降。3TC与ASODN联用对HBeAg的抑制呈拮抗、相加和协同作用,其拮抗作用随着3TC浓度的增加逐渐减弱。ADV与ASODN联用对HBeAg的抑制只呈相加作用。3TC和ADV分别与ASODN联用对HBV DNA的抑制均表现为相加作用或协同作用,取ADV0.5μmol/L与取ASODN0.2μmol/L联用时对HBV DNA的抑制率可提高到72.6%。ASODN在一定条件下分别与3TC及ADV联用有相加或协同抗HBsAg、HBeAg及HBV DNA的表达,尤以抗HBsAg作用显著增强。     

反义前S／S基因转移表达抗乙型肝炎病毒的研究

为了观察反义基因转录表达抗乙型肝炎病毒（HBV）的作用,将HBVayw前S／S基因（PreS／S）片段反向插入逆转录病毒载体质粒,构建preS／S反义基因重组体,经转染PA317细胞后,获得重组逆转录病毒颗粒。用重组病毒转导2．2．15细胞后,第3天即可见细胞培养上清HBV表面抗原（HBsAg）和e抗原（HBeAg）表达量减少,到转导后第5天,细胞培养上清中的HBsAg和HBeAg表达量降到最低水平,HBsAg抑制率为71％,HBeAg抑制率为27％,抑制作用至少可持续到转导后第11天。空载及正向插入基因的重组逆转录病毒转导对2,2．15细胞内HBV抗原表达无抑制作用。     

反义前C／C基因转移表达抗乙型肝炎病毒作用的研究

为了观察逆转录病毒载体包装细胞系统介导反义基因转移表达的抗乙型肝炎病毒（HBV）作用,将HBVayw前C／C基因（preC／C）片段反向插入逆转录病毒载体质粒。将重组体转染逆转录病毒包装细胞PA317后,获得重组逆转录病毒。用重组逆转录病毒感染2．2．15细胞后发现,感染后第3天,细胞培养上清HBV表面抗原（HBsAg）和e抗原（HBeAg）表达量即明显减少,抑制作用于感染后第5天达到高峰,其中对HBsAg表达的抑制率为27．0％,对HBeAg表达的抑制率为59.5％。反义基因重组逆转录病毒感染2.2．15细胞对HBV抗原表达的抑制作用至少可以持续到转导后的第11天。空载及正义基因重组逆转录病毒感染对2．215细胞HBV抗原表达无明显抑制作用。此外,反义基因重组逆转录病毒感染对2.2.15细胞HBVDNA复制也有抑制作用．无细胞毒性。     

HBV prec/c shRNA慢病毒真核表达载体的构建和鉴定

目的构建针对HBV prec/c区的shRNA真核表达载体psiHBV,感染HepG2 2.15细胞后观察其对HBeAg表达的抑制作用,为探讨防治HBV感染的新措施提供实验依据。方法针对HBV prec/c基因序列,构建shRNA表达载体psiHBV1、psiHBV2和无关序列psiHBVc。psiHBV与慢病毒辅助系统质粒共转染293T细胞组装慢病毒颗粒后,感染HepG2 2.15细胞,RT-PCR检测prec/c mRNA的转录,微粒子化学发光分析仪(MEIA)检测细胞上清和细胞裂解液中HBeAg表达。结果重组质粒双酶切和测序鉴定与预期结果相符合;组装慢病毒颗粒感染HepG2 2.15细胞后,prec/c mRNA转录降低;与对照组比较,HBeAg的表达水平也显著降低,病毒颗粒对HBeAg表达的抑制作用差异有统计学意义(P<0.01)。结论成功构建针对HBVprec/c的慢病毒载体psiHBV1、siHBV2,慢病毒介导的RNA i能抑制HBV表达,为应用RNA干扰技术治疗乙型肝炎提供了实验依据。     

pRNA介导的RNA干扰抑制HBV表达和复制的研究

为了研究由pRNA携带的siRNA(HBVsi18-42)所介导的RNAi过程能有效地抑制HBV的基因表达和病毒复制,我们利用细胞模型和高压注射小鼠模型评价HBVsi18-42对HBV复制和基因表达的抑制作用.通过Western印迹检测细胞内的HBsAg含量,用ELISA检测细胞培养上清和小鼠血清中的HBsAg水平,采用Southern印迹检测HBV的复制中间体,通过免疫组织化学检测肝组织切片中HBcAg的表达情况.试验结果显示,HBVsi18-42能以剂量依赖的方式在293T细胞中抑制HBsAg的表达以及在HepG2细胞中下调病毒HBsAg和HBeAg的表达和病毒复制中间体的水平.在小鼠模型中,注射后的3d内HBVsi18-42使小鼠血清中HBsAg的水平分别下降了98.98%、77.07%和60.73%,免疫组织化学检测显示,在注射后的第3天小鼠肝组织内HBcAg阳性细胞数减少了79.1%.初步结果显示HBVsi18-42无论是在细胞或是在小鼠模型中都能下调HBV的复制和基因的表达.本研究为我们下一步实现由pRNA介导的靶向RNAi及基因治疗提供了理论和技术支持.     

转录因子Sp1上调前列腺癌细胞中CD59的表达

目的:构建针对Sp1基因siRNA真核表达载体,转染前列腺癌细胞PC-3,研究反式作用因子Sp1时CD59表达的影响.方法:应用siRNA表达载体介导的RNAi技术,构建含特异性sp1基因的重组载体pSUPER-siSp1,脂质体法转染前列腺癌细胞,G418筛选建立稳定表达转染基因的细胞株,Western blotting检测转染细胞中sp1和CD59基因的表达,MTT和染料释放试验判断CD59基因抑制后对补体溶破的抵抗作用.结果:成功构建了Sp1基因siRNA真核表达载体,转染PC-3细胞可表达荧光蛋白,稳定转染的Pc-3细胞Sp1及CD59基因蛋白水平降低,MTT和染料释放实验表明CD59基因受抑制后对补体溶破的抵抗作用降低.结论:siRNA-Sp1重组载体有效地抑制了CD59的表达,降低CD59的抗补体活性,结果证明反式作用因子Sp1是CD59表达调控中重要的转录因子,为探讨CD59在肿瘤细胞中高表达的研究奠定了基础.     

Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.     

TPP1基因慢病毒干扰载体的构建及鉴定

目的:构建抑制TPP1基因的短发夹RNA(sh RNA)干扰载体。方法:以人的TPP1基因为靶序列,设计并合成sh RNA序列TPP1-si1和TPP1-si2,分别与RNA干扰慢病毒载体pll3.7连接,双酶切鉴定质粒得到阳性克隆pll-TPP1-si1和pll-TPP1-si2,经测序正确后将其与慢病毒包装载体(RRE、REV、VSVG)共转染293T细胞,进行病毒的包装,将得到的病毒感染稳定高表达外源TPP1蛋白的HT1080细胞,通过Western印迹检测其对TPP1蛋白表达的抑制效果,并进行比较;将抑制效果好的病毒感染高表达外源Pot1蛋白的Hep G2细胞,检测Pot1蛋白在内源TPP1被敲低的情况下能否在端粒定位。结果:经双酶切验证,外源片段成功插入载体pll-TPP1-si1和pll-TPP1-si2中;pll-TPP1-si1和pll-TPP1-si2均能明显抑制TPP1的表达,其中pll-TPP1-si1的抑制效果最好;pll-TPP1-si1病毒感染高表达外源Pot1蛋白的Hep G2细胞,经免疫荧光鉴定能够有效抑制内源TPP1的表达,使Pot1不再端粒定位。结论:构建的抑制TPP1基因的sh RNA干扰载体能有效抑制TPP1的表达,为端粒蛋白TPP1功能的研究奠定了实验基础。     

Two approaches to construct mammalian expression vector of shRNA to reduce expression and replication of HBV in vitro

Two approaches have been developed to construct plasmids that mediate RNA interference to inhibit the replication and expression of HBV in 2.2.15 cell. The overlapping PCR extension and restriction enzyme-digestion were used to generate DNA fragments encoding designed shRNA based on sequences of ORF C of HBV genome. The pU6 derived vectors were constructed to develop plasmid based shRNA delivery systems termed pU6/HBVi. There were significant reductions in the expression of HBsAg and HBeAg between cells transfected with pU6/HBVi and control groups (as to HBsAg: P < 0. 01; and HBeAg: P < 0. 01). Consistently, the HBV DNA copies were reduced from 2.71 x 10(7) to <5 x 10(2) copies with or without pU6/HBVi. These results suggested that shRNA delivery by recombinant plasmids harboring shRNA encoding DNA fragment of interest generated either by overlapping PCR extension or restriction enzyme-digestion, could inhibit expressions of viral proteins and reduce viral replications. The pU6 derived plasmids might be a useful shRNA delivery system in mammalian cells. In addition, we found siRNA based on stealth 2311 was a potent RNAi target of HBV genome.     

人Hes1-shRNA，Hes5-shRNA慢病毒表达载体的构建、包装及鉴定

目的构建人Hesl-shRNA和Hes5-shRNA慢病毒表达载体,为Notch—Hes信号通路的相关研究奠定基础。方法根据人Hes1,Hes5基因mRNA序列分别设计、合成多对互补的DNA单链寡核苷酸,退火后克隆至pENTR／U6入门载体。通过入门载体瞬时转染神经胶质瘤U251细胞筛选有效干扰序列。将含有效干扰序列的入门载体与pLenti6／BLOCK—iT—DEST载体进行LR重组构建Hesl—shRNA和Hes5-shRNA慢病毒表达载体,经脂质体介导入293FT细胞,包装成慢病毒。用该慢病毒感染U251细胞,Western印迹法分别检测Hes1,Hes5蛋白的表达。结果分别构建了针对Hes1和Hes5基因的特异性shRNA慢病毒表达载体,其包装获得慢病毒可有效感染U251细胞并分别对HeM,Hes5蛋白的表达有显著抑制作用。结论成功构建了Hesl—shRNA和Hes5-shRNA慢病毒表达载体。     

绿色荧光蛋白标记的TGF-β1shRNA真核表达载体的构建及鉴定

目的：构建小鼠转化生长因子β1（TGF-β1）短发夹RNA（shRNA）真核表达载体,探讨TGF-β1在血管发育中的调控作用。方法：根据GenBank小鼠TGF-β1mRNA序列,设计合成三对短链寡核苷酸,退火后形成双链DNA并克隆至入门载体DEN_mH1c。将插入目的基因片段的入门载体与带有绿色荧光蛋白（GFP）标签的shRNA真核表达载体pDS_hpEy进行LR重组反应,完成三个TGF-β1shRNA表达载体的构建,分别命名为pDS_Ta,pDS_Tb和pDS_Tc。经测序确认后,转染小鼠成纤维细胞（NIH／3T3）,筛选稳定表达的细胞克隆,以RT-PCR及Westem blot方法检测转染后TGF-β1mRNA和蛋白表达。结果：RT-PCR和Western blot显示pDS_Tc可明显下调NIH／313细胞TGF-β1的mRNA和蛋白表达,mRNA下调约为70％,蛋白表达减少约65％。结论：GFP标签TGF-β1shRNA表达载体能够阻断TGF-β1基因表达,可作为研究TGF-β1调控血管发育机制的一个工具,为阐明TGF-β1信号传导通路奠定基础。     

Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors

BACKGROUND: There has been much research into the use of RNA interference (RNAi) for the treatment of human diseases. Many viruses, including hepatitis B virus (HBV), are susceptible to inhibition by this mechanism. However, for RNAi to be effective therapeutically, a suitable delivery system is required. METHODS: Here we identify an RNAi sequence active against the HBV surface antigen (HBsAg), and demonstrate its expression from a polymerase III expression cassette. The expression cassette was inserted into two different vector systems, based on either prototype foamy virus (PFV) or adeno-associated virus (AAV), both of which are non-pathogenic and capable of integration into cellular DNA. The vectors containing the HBV-targeted RNAi molecule were introduced into 293T.HBs cells, a cell line stably expressing HBsAg. The vectors were also assessed in HepG2.2.15 cells, which secrete infectious HBV virions. RESULTS: Seven days post-transduction, a knockdown of HBsAg by approximately 90%, compared with controls, was detected in 293T.HBs cells transduced by shRNA encoding PFV and AAV vectors. This reduction has been observed up to 5 months post-transduction in single cell clones. Both vectors successfully inhibited HBsAg expression from HepG2.2.15 cells even in the presence of HBV replication. RT-PCR of RNA extracted from these cells showed a reduction in the level of HBV pre-genomic RNA, an essential replication intermediate and messenger RNA for HBV core and polymerase proteins, as well as the HBsAg messenger RNA. CONCLUSIONS: This work is the first to demonstrate that delivery of RNAi by viral vectors has therapeutic potential for chronic HBV infection and establishes the ground work for the use of such vectors in vivo.     

LRP16短发夹RNA慢病毒载体的构建及LRP16稳定抑制细胞的建立简

目的：构建靶向LRPl6基因的短发夹RNA（shRNA）慢病毒表达载体,鉴定其在HeLa细胞中对LRP16的抑制效果。方法：构建pWPT-U6-LRPl6shRNA-CMV-GFP慢病毒载体,通过病毒感染、细胞筛选、Western印迹等步骤,获得LRP16基因稳定抑制的细胞株。结果：构建了具有LRP16干扰效果的慢病毒载体,感染HeLa细胞后获得了稳定沉默LRP16及对照的细胞株;经克隆筛选,在荧光显微镜下观察到近似100％感染细胞发出绿色荧光;Western印迹证实pWPT-U6-L374-CMV-GFP和pWPT-U6-L668-CMV-GFP均可显著抑制HeLa细胞株中LRP16蛋白的表达,其中pWPT-Gsi-L374-GFP的抑制效果更好。结论：构建了靶向人LRP16基因shRNA慢病毒载体及LRP16稳定抑制的HeLa细胞系。