靶向X区的siRNA抑制乙型肝炎病毒基因的表达和复制

目的构建针对乙型肝炎病毒（HBV）X基因的siRNA表达载体，观察其对HBV基因表达和复制的影响。方法设计并合成针对HBVX区基因的siRNA寡核苷酸，经退火形成双链后克隆人pSUPER载体，构建成功的siRNA表达载体与pTK-Hyg质粒共转染稳定表达HBV的HepG22．2．15细胞，潮霉素抗性筛选获得稳定细胞克隆，对所得细胞培养上清液中的HBsAg和HBeAg进行定量检测，逆转录-聚合酶链反应（RT-PCR）检测靶基因mRNA的抑制效果，荧光定量PCR检测HBVDNA。结果成功构建了针对HBVX基因的siRNA表达载体pSUPER-X1和pSUPER-X2，两种siRNA均能明显抑制HepG22．2．15细胞的HBsAg和HBeAg分泌，抑制率分别为97％和88％，RT-PCR结果显示HBV的mRNA表达降低，荧光定量PCR结果证实siRNA能降低HBVDNA拷贝数2个数量级。结论载体产生的针对HBVX基因的siRNA能高效、特异地抑制HBV基因的表达和复制。     

转基因细胞2．2．15的培养及分泌表达的动力学变化

目的 为了筛选抗乙型肝炎病毒(HBV)药物的细胞模型。本研究探索了转基因细胞2．2．15的培养条件及分泌表达的动力学变化。方法 用ELISA法动态检测2．2．15细胞分泌表达HBsAg和HBeAg的动力学变化。用PCR技术检测细胞和细胞培养上清中HBV-DNA。结果 2．2．15细胞在适宜的培养条件下能够持续分泌表达HBsAg和HBeAg，培养第6天达分泌高峰。第7天有所下降。PCR结果证明细胞和培养液上清中HBV-DNA为阳性。结论 转基因细胞2．2．15可作为筛选抗HBV药物的细胞模型。     

HBV感染与IL 29表达的相关性

摘要：目的：检测乙型肝炎患者血清中Ⅲ型干扰素IL-29的表达水平,探讨HBV感染是否会上调IL-29的表达。 方法：收集健康人、乙肝患者的血清,用ELISA试剂盒检测IL-29的表达,并从新鲜全血分离外周血单个核细胞（PBMC）,提取总RNA,半定量PCR方法检测IL-29 mRNA表达水平。构建带萤光素酶（luciferase）报告系统的IL-29启动子质粒,分别转染到能稳定表达HBV病毒颗粒的HepG2.2.15 细胞与对照的HepG2细胞,测萤光素酶活性,并分析HBV对IL-29启动子活性的影响。 结果: 乙肝患者血清中IL-29与IL-29 mRNA的表达水平显著高于健康人,HBeAg阳性患者血清中IL-29及IL-29 mRNA的表达水平均高于HBeAg阴性患者。转染至HepG2.2.15 细胞中的IL-29启动子报告质粒的活性显著高于转染至对照HepG2细胞者。 结论：HBV感染后,激活机体Ⅲ型干扰素IL-29的启动子活性,从而使IL-29的表达水平显著上调。     

短发夹RNA对肝癌细胞细胞间粘附分子-1基因表达的影响

目的利用小片段干扰RNA（siRNA）在肝癌细胞内诱导RNA干扰（RNAi），抑制细胞间粘附分子-1（ICAM-1）基因表达，在体外进行RNAi对肝癌治疗的实验研究。方法根据ICAM-1基因设计shRNA片断，构建pGenesil-1／ICAM-1表达载体，转入E．coli菌，并用Lipofectamine tm2000转染HepG2细胞，通过MTT方法检测细胞活性状态，应用免疫组织化学的方法检测肝癌细胞中ICAM-1基因蛋白质表达抑制情况。结果本实验已成功构建了针对ICAM-1基因的pGenesil-1／ICAM-1质粒，免疫组织化学结果显示：RNA干扰（RNAi）能特异而有效地抑制HepG2细胞中ICAM-1基因的表达。结论构建的pchlesd-1／ICAM-1重组质粒能有效抑制ICAM-1基因在肝癌细胞株HepG2中的表达和瘤细胞增殖。     

乙型肝炎病毒rtA181V/T和rtN236T变异阿德福韦耐药株表达质粒的构建及其病毒学特征

目的构建乙型肝炎病毒阿德福韦耐药变异株（rtA181T／V和rtN236T）表达载体并对其体外复制能力和耐药性等病毒学特征进行体外研究。方法以含1．2倍拷贝HBV DNA全基因的质粒PUC-HBV1．2WT为模板，PCR定点诱变技术构建含阿德福韦耐药株（rtA181V／T和rtN236T）的目的质粒，测序验证并利用变异株特异检测引物进行PCR检测；转染人肝癌细胞系HepG2，ELISA检测上清中分泌的HBsAg和HBeAg水平，荧光定量PCR检测上清中病毒DNA水平，Southern blotting检测胞浆HBV复制中间体水平，比较野生株和变异株体外复制能力和对阿德福韦敏感性的差异。结果构建的rtA181T、rtA181V和rtN236T表达质粒可用于变异株特异检测引物检测相应变异的阳性对照品；转染HepG2细胞后均可获得高水平的病毒抗原表达，胞浆和细胞培养上清中可以检测到HBV复制中间体和病毒颗粒的存在；三种变异均可单独导致对阿德福韦耐药，IC50为野生株的2．8至4．7倍；体外复制能力较野生株降低，分别为野生株的94．2％、89．0％和77．7％。结论成功构建乙型肝炎病毒阿德福韦变异株表达质粒并应用于变异株特异引物扩增检测技术，体外实验证实rtA181V／T和rtN236T单独变异均可导致HBV对阿德福韦耐药，且变异株的病毒复制能力较野生株有所下降。     

HBV核心蛋白拮抗α干扰素抗病毒活性的初步研究

目的 探讨HBV核心蛋白（HBc）对α干扰素（IFN-α）抗病毒活性可能存在的拮抗机制。 方法 以表达抗黏液病毒A蛋白(MxA)的重组质粒pcDNA3.1-Flag-MxA （野生型）转染肝胚瘤细胞株HepG2.2.15细胞,用ELISA和real-time PCR法分别检测转染后HepG2.2.15细胞上清HBsAg、HBeAg和细胞外HBV DNA。HBc核心蛋白表达质粒（pHBc-EGFP）转染HepG2细胞后,RT-PCR法分析IFN-α抗病毒蛋白MxA、双链RNA激活的蛋白激酶（PKR）和2′,5′-寡腺苷酸合成酶（2′,5′-OAS）等mRNA水平。结果 转染MxA重组质粒的HepG2.2.15细胞能够表达MxA,转染48 h时HBsAg、HBeAg比空白对照组显著减少（P＜0.05）,HBV DNA无显著性差异。转染pHBc-EGFP的HepG2细胞,经1 000 IU/ml IFN-α处理后,抗病毒蛋白PKR和2′,5′-OAS mRNA水平未见明显改变,而MxA-mRNA水平显著降低（P＜0.05）。 结论 HBV核心蛋白可能通过抑制抗病毒蛋白MxA的表达,而发挥对IFN-α的拮抗作用。     

环指蛋白6对胰岛素受体底物1泛素化作用的研究

目的：构建环指蛋白6（RNF6）真核表达载体,并探讨其对胰岛素受体底物1（IRS-1）的泛素化作用。方法以人cDNA为模板,PCR 扩增 RNF6全长编码基因,并将其克隆至载体质粒 pcDNA3．1-CHA 中,将重组质粒转染肝癌细胞株 HepG2,利用实时荧光定量 PCR（RT-PCR）、免疫印迹（Western blot）检测细胞内 IRS-1 mRNA 水平及其蛋白表达水平。结果携带RNF6目的基因的质粒转染 HepG2细胞48 h 后 IRS-1的 mRNA 表达水平降低,为对照组的69％,显著低于对照组,差异有统计学意义（P ＜0．01）。结论RNF6引起 IRS-1的表达下调,这一泛素化过程可能导致胰岛素信号转导障碍。     

HSPC238对肝癌细胞中RB基因表达的影响

目的观察HSPC238对肝癌细胞中RB mRNA和pRb蛋白水平的影响。方法将PcDNA3.1-HSPC238质粒和PLL3.7-HSPC238干扰载体分别转染HepG2细胞,用实时荧光定量PCR的方法检测RB mRNA的表达量;采用Western blotting法检测pRb蛋白的表达。结果和对照组相比,转染高表达HSPC238载体时,HepG2细胞中RB的mRNA水平和pRb表达量也增加;相反地,和对照组相比,转染低表达HSPC238载体时,HepG2细胞中RB的mRNA水平和pRb蛋白的表达量也减少。以上结果差异均有统计学意义(P0.05)。结论在HepG2细胞中HSPC238对RB基因的表达存在正向调控作用。     

HBV前S2抗原、前S2抗体与其病毒标志物的关系

目的：探讨乙型肝炎患者S2抗原,抗体与HBV M,HBV－DNA之间关系。方法：对146例乙型肝炎患者用ELISA法检测前S2抗原,前S2抗体及乙型肝炎病毒标志物（HBV M）;用聚合酶链反应（PCR）法检测乙型肝炎病毒HBV－DNA。结果：急性肝炎,慢性肝炎,肝硬变患者前S2抗原检出率分别为8．33％,77．07％;前S2抗体的检出率分别为75％,9．34％,3．7％,前S2抗原在HBV－DNA,HBeAg阳性病例中检出率明显高于阴性组（P＜0．001）,前S2抗体阳性病例HBV－DNA,HBeAg均为阴性。结论：前S2抗原的检出意味着病毒有复制或有传染性,前S2抗体的出现标志着病毒被清除,在慢性肝病中检出并不意味病情稳定,同时发现该抗体可以在急性乙型肝炎急性期及血清HBV M全部阴性的患者中检出。     

解偶联蛋白-2 RNA干扰慢病毒载体的构建

目的:构建解偶联蛋白-2(UCP-2)基因的RNA干扰慢病毒载体.方法:根据RNA干扰序列设计原则,针对UCP-2mRNA(NM_011671)设计3个RNA干扰靶点序列,合成含干扰序列的DNA双链,酶切后连入慢病毒转移质粒pGCL-GFP中.连接产物转化感受态细胞,所获克隆行PCR鉴定和序列测定.构建成功的RNA干扰质粒与UCP-2表达质粒共转染293T细胞,荧光显微镜下观测转染效果,Western blot检测UCP-2蛋白表达,筛选出干扰效果最好RNA干扰质粒.将该质粒与两种辅助包装原件载体质粒pHelper 1.0(gag/pol元件)载体,Helper 2.0(VSVG元件)共转染293T细胞包装成慢病毒并检测滴度.结果:PCR和测序结果显示针对3个靶点的RNA干扰质粒均构建成功;通过Western blot检测获得最优干扰靶点,并成功包装成滴度为5×108TU/ml的慢病毒.结论:成功构建可供感染的解偶联蛋白-2 RNA干扰慢病毒载体,为后续靶向抑制该基因表达以提高脂肪肝移植成功率的研究莫定物质基础.     

The short hairpin RNA driven by polymerase II suppresses both wild-type and lamivudine-resistant hepatitis B virus strains

BACKGROUND: Chronic infection with hepatitis B virus (HBV) is widespread because of the limited availability of therapeutic treatments. Although previous reports have suggested that RNA interference has promise as a treatment for HBV infection, further studies of long-term and off-target drug effects on HBV, especially on drug-resistant strains of HBV, are needed. Therefore, seven vectors that express short hairpin RNAs (shRNAs), driven by the polymerase II promoter, pSilencer4.1/HBV, were constructed to target open reading frames (ORFs) of the HBV C and S genes from wild-type and drug-resistant strains. Treatment efficiency was also assessed. METHODS: The pSilencer4.1/HBV vectors were investigated in HepG2.2.15 cells and transgenic mice that consistently produce wild-type HBV. Additionally, vectors that produce a lamivudine-resistant strain of HBV were developed and cotransfected, along with pSilencer/HBV, into both HepG2 cells and mice. The effects of polymerase-II-driven pSilencer4.1/HBV were compared with those of polymerase-III-driven pSilencer3.1/HBV at both the gene and protein level. RESULTS: pSilencer4.1/HBV inhibited the expression of viral protein, DNA and HBV subtype ayw mRNA in both HepG2.2.15 cells and transgenic mice. Toxicity, as well as off-target effects, did not occur after a short- to medium-term examination. Moreover, an HBV strain resistant to lamivudine, subtype adr, was suppressed by shRNA in both HepG2 cells and mice. In contrast to polymerase III, vectors that used polymerase II could drive efficient silencing without off-target effects. CONCLUSIONS: Silencing by shRNA dramatically inhibited HBV expression and replication regardless of strain type. ShRNA could therefore be a promising treatment for HBV infection.     

反义锁核酸体外抗乙肝病毒的实验研究

目的探讨锁核苷酸(LNA)修饰的反义寡核苷酸抑制乙型肝炎病毒(HBV)表达的效果及其抗HBV作用的特点。方法针对HBV S基因同一靶位分别合成三段不同化学修饰的反义寡核苷酸:锁核酸修饰、未修饰和全硫代修饰,并以无关序列为对照直接作用于HepG22.2.15细胞,ELISA法动态检测细胞上清液中HBsAg含量;实时荧光定量PCR法(FQ-PCR)检测细胞上清中HBV DNA含量;MTT法监测细胞活性。结果针对HBV S基因的反义LNA能显著抑制HepG22.2.15细胞表达HBsAg(P<0.05)且第3天出现抑制高峰,抑制作用随时间延长逐渐减弱。反义锁核酸组对HBsAg及细胞内、细胞外HBV DNA均有较强抑制作用,最高抑制率分别为51.19%、56.73%、86.9%,与其他实验组相比差异有统计学意义(P<0.05),与对照组比较差异有统计学意义(P<0.01);LNA在20μmol/L浓度范围内对细胞代谢无影响。结论针对S基因的反义LNA体外能发挥高效、特异、低毒的抗HBV作用,为乙型肝炎的基因治疗注入了新的思路。     

乙型肝炎病毒对胰岛素样生长因子结合蛋白7表达的影响及其临床意义

目的探讨乙型肝炎病毒（HBV）对胰岛素样生长因子结合蛋白7（IGFBP7）表达的影响及其临床意义。方法逆转录-聚合酶链反应（PCR）检测IGFBP7mRNA的表达水平,酶联免疫吸附试验（ELISA）检测血清IGFBP7含量,分析IGFBP7在慢性乙型肝炎（CHB）、肝纤维化（LC）和肝细胞癌（HCC）患者间血清含量的差异。结果IGFBP7mRNA在HepG2．2．15细胞中的表达水平较HepG2高;与健康对照组相比,HBV感染者IGFBP7血清学水平明显升高（P〈0．05）;IGFBP7在Lc和HCC患者低于CHB患者（P〈0．05）。结论HBV能够上调IGFBP7的表达,其血清水平与疾病进程呈负相关。     

Long-term suppression of hepatitis B virus replication by short hairpin RNA expression using the scaffold/matrix attachment region-based replicating vector system pEPI-1

Since the emergence of viral resistance of hepatitis B virus (HBV) during treatment is becoming an important issue even with newer drugs, there is a need for alternative treatment options such as, for example, RNA interference (RNAi) technology. While short-term suppression of HBV replication is easily achieved with small interfering RNA oligonucleotides, this is not the case for long-term suppression due to the lack of an optimal vector system. Based on the nonviral scaffold/matrix attachment region (S/MAR)-based vector system pEPI-1, which is free of common side effects and is stably retained as an episome even in the absence of selection, we designed a short hairpin RNA (shRNA) expression vector called pEPI-RNAi for HBV suppression. HBV-replicating HepG2.2.15 cells were transfected with pEPI-RNAi, and the intracellular status of the plasmid was followed by PCR and Southern analysis. HBV replication was measured on the DNA, RNA, and protein level. HBV RNA expression was reduced by almost 85% 3 months posttransfection with pEPI-RNAi. At 8 months posttransfection in the absence of antibiotic selection pressure, the suppression level was still 70% and the vector was retained as an episome. The reduction of total intracellular HBV DNA at this point was 77%, showing a marked suppression of HBV DNA replication. At a comparable level, secretion of viral antigens, as well as progeny HBV virions, was inhibited. The S/MAR-based vector system pEPI-1 allows long-term suppression of HBV replication by the expression of suitable shRNAs. Due to its unique properties compared to commonly used vectors, it provides an interesting option for the treatment of chronically HBV-infected individuals.     

N-乙酰半胱氨酸体外抗乙肝病毒作用

目的：探讨NAC体外抗乙肝病毒活性及其机制。方法：体外以转染了乙肝病毒的HepG2-2．2．15细胞株作为实验对象，培养细胞中加入不同浓度的NAC(0，3，10，30mmol／L)。用酶联免疫吸附法(ELISA)测定培养上清液中的乙肝表面抗原(HBsAg)、e抗原(HBeAg)。半定量PCR测定细胞内及培养上清液中的HBV DNA的变化，RT-PCR分析细胞内HBV表面抗原mRNA的变化。结果：NAC对HBsAg、HBeAg均有抑制作用，但NAC对HBsAg的抑制较HBeAg强，抑制率达90％左右。且抑制作用具有剂量和时间依赖性。半定量PCR结果显示细胞内的HBV DNA含量随着NAC浓度的增加而升高，而培养上清中的HBV DNA的含量降低。RT-PCR结果表明细胞内HBsAg mRNA没有显著变化。结论：NAC体外具有抗HBV活性，其抑制作用发生在转录后水平。     

Evidence for dual effects of DNA-reactive bile acid derivatives (Bamets) on hepatitis B virus life cycle in an in vitro replicative system

A liver targeting strategy to direct antiviral drugs toward hepatitis B virus (HBV) was investigated. As model drugs we used cisplatin-bile acid derivatives (Bamets) to determine the production of virions by HBV-transfected hepatoblastoma cells (HepG2 2.2.15). Drug uptake was determined using flameless atomic absorption spectrometry to measure platinum cell contents. Cytotoxic effect was determined by formazan formation and neutral red uptake tests. The release of viral surface protein was evaluated by ELISA. The abundance of HBV-DNA in the medium was determined by quantitative real-time PCR and its structure by Southern blot analysis. The uptake of Bamets by HepG2 2.2.15 cells was higher than that of cisplatin. At concentrations lower than 10 microM, distinct Bamets have no toxic effect on host cells, whereas cisplatin dramatically reduced cell viability at concentrations higher than 1 microM. All the drugs tested inhibited the release of viral proteins to the medium, but induced a marked and progressive dose-dependent increase in the amount of viral DNA in the medium. This was mainly due to the release of short fragments of HBV-DNA in the case of cisplatin. On the contrary, Bamets induced an enhanced release of circular forms of HBV-DNA. These findings suggest the existence of a dual effect of Bamets on HBV life-cycle by enhancing the production of DNA replicative intermediates but reducing the secretion of complete virions. Altogether these characteristics recommend consideration of these compounds as a useful experimental tool in the investigation of novel liver targeted therapeutic agents based on bile acid derivatives for the treatment of HBV infections, or to carry out further studies on the HBV life cycle.     

他克莫司在体外对HepG2.2.15细胞增殖及乙型肝炎病毒复制的影响

背景:肝癌复发与乙肝病毒再感染两者关系尚不明确,有人认为与肝移植后免疫抑制剂应用有关.目的:观察他克莫司在体外对肝癌HepG2.2.15细胞增殖及对细胞内乙肝病毒复制的影响.方法:选择肝癌HepG2.2.15细胞进行体外培养,第3代细胞培养24 h后加他克莫司进行干预,0 g/L他克莫司作为对照组,50 g/L 他克莫司为低浓度组,100,500 g/L他克莫司为中浓度组,1 000,3 000 g/L他克莫司为高浓度组.结果与结论:①中、高浓度的他克莫司对HepG2.2.15细胞有增殖抑制作用,低浓度无抑制作用,且有相关性.②高浓度他克莫司作用时使HepG2.2.15细胞停止在G0/G1期.③他克莫司可以使HepG2.2.15细胞中CyclinA表达降低,且呈浓度依赖性,他克莫司浓度越高,CyclinA表达越少.④他克莫司作用HepG2.2.15细胞,对HBV的复制无影响.结果说明他克莫司在体外对HepG2.2.15增殖有抑制作用,其中CyclinA可能发挥一定的作用,而对乙肝病毒复制没有影响.