应用RNA干扰抑制乙型肝炎病毒复制和表达

RNA干扰(RNAi)是一种由双链RNA(dsRNA)引发的序列特异性基因沉默机制。在这一转录后基因沉默(PTGS)过程中,与双链RNA有同源序列的单链RNA(如mRNA)被降解,从而阻断基因表达。RNAi过程的关键是由双链RNA     

乙醇对乙型肝炎病毒转基因小鼠病毒复制和基因表达的影响

乙型肝炎病毒(HBV)在体内的持续感染、复制和基因表达是急、慢性乙型肝炎发病机制的重要环节。嗜酒明显影响乙型肝炎的预后，使其易于重型化、慢性化，且肝癌发生率升高。嗜酒与HBV感染对肝脏损伤有协同作用。本研究旨在通过对HBV转基因小鼠的乙醇干预，探讨它对HBV复制和基因表达的影响及其机制，以利于对乙型肝炎及酒精相关性肝病的防治。     

靶向HBV C区RNAi对HBV复制和HBeAg表达的抑制作用

目的观察靶向HBVC基因区的RNA干扰（RNAi）对HBV复制的抑制作用。方法选择HBV基因组C区的Nt2021-2049作为靶序列,合成相应的正、反义寡核苷酸,退火后形成双链,克隆人shRNA表达质粒,将得到的质粒与HBV质粒共转染HepG2细胞,观察HepG2细胞中HBV的受抑情况。结果病毒复制及HBeAg的表达明显受到抑制。结论所选靶区通过RNAi可有效抑制HBV复制。     

HBV S基因的反义锁核酸对乙型肝炎转基因小鼠HBV复制和表达的影响

目的: 探讨乙型肝炎病毒(hepatitis B virus,HBV)S基因的反义锁核酸(LNA)对乙型肝炎转基因小鼠HBV复制和表达的影响.方法: 将30只HBV转基因小鼠随机分为5组,每组6只. 第1组为5% GLU液对照组, 第2组为空脂质体对照组, 第3组为单LNA组, 第4组为S-ASODN脂质体组, 第5组LNA脂质体组. 反义LNA经尾静脉注入小鼠体内, 采用ELISA法检测血清HBsAg;PCR定量检测血清HBV DNA含量;免疫组织化学法检测肝细胞HBsAg的表达;自动生化分析仪检测ALB、ALT、BUN、CR、ApoA1、ApoB等指标;小鼠肝脏、肾脏做常规病理切片HE染色, 观察反义LNA对小鼠脏器的影响.结果: 注射反义LNA 1 d、3 d、7 d、14 d后,LNA-脂质体组对血清HBsAg的表达抑制率分别为41.7%、52.8%、57.8%及30.5%. 对HBV DNA的抑制率分别为18.5%、36.1%、52.9%和32.7%, 与对照组比较均有显著性差异(P <0.05);全自动生化分析仪检测血清中ALB、ALT、BUN、CR、ApoA1、ApoB等指标, 各组结果与对照组比较均无显著性差异(P >0.05);小鼠肝细胞HBsAg的表达显著低于对照组. HE染色显示小鼠肝肾功能及组织学未见异常.结论: HBV S基因反义LNA对乙型肝炎病毒转基因鼠HBV复制和表达有显著抑制作用.     

不同靶区RNA干扰抑制乙型肝炎病毒复制与表达

目的 观察针对乙型肝炎病毒（HBV）不同靶区发夹样RNA（shRNA）抑制HBV复制与表达的效率。方法 根据shRNA表达载体设计原则，设计并构建了针对HBVP、C、S和X区7种特异性和2种序列突变的shRNA表达载体，将其与1．3倍HBV全基因表达载体共转染于HepG2细胞，通过Southernblot和酶联免疫吸附法分别检测HBV DNA复制中间体和乙型肝炎表面抗原（HBsAg）水平，来观察shRNA抗HBV复制与表达的效率。结果 发现针对HBV不同靶区shRNA均有抗HBV复制和表达效果，以P1、S2、C2、S1和X抑制HBV DNA复制效率较高，P1的抑制率高达95％。HBsAg表达受抑制情况与HBV DNA复制受抑制的程度相似，其中C2、C1和S2对HBsAg的抑制作用较强。结论 针对HBV四个开放性读码框进行RNA干扰对HBV复制和抗原表达均有抑制作用，其中P1和C2分别对HBV复制和表达的抑制效率较高。     

DDX3-mediated miR-34 expression inhibits autophagy and HBV replication in hepatic cells

HBV entry to the host cells and its successful infection depends on its ability to modulate the host restriction factors. DEAD box RNA helicase, DDX3, is shown to inhibit HBV replication. However, the exact mechanism of inhibition still remains unclear. DDX3 is involved in multitude or RNA metabolism processes including biogenesis of miRNAs. In this study, we sought to determine the mechanism involved in DDX3-mediated HBV inhibition. First, we observed that HBx protein of HBV downregulated DDX3 expression in HBV-infected cells. Overexpression of DDX3 inhibited HBx, HBsAg and total viral load, while its knockdown reversed the result in Hep G2.2.15 cells. Expression of miR-34 was downregulated in HBV-infected cells. Overexpression of pHBV_1.3 further confirmed that HBV downregulates miR-34 expression. Consistent with the previous finding that DDX3 is involved in miRNA biogenesis, we observed that expression of miR-34 positively corelated with DDX3 expression. miRNA target prediction tools showed that miR-34 can target autophagy pathway which is hijacked by HBV for the benefit of its own replication. Indeed, transfection with miR-34 oligos downregulated the expression of autophagy marker proteins in HBV-expressing cells. Overexpression of DDX3 in HBV-expressing cells, downregulated expression of autophagy proteins while silencing of DDX3 reversed the results. These results led us to conclude that DDX3 upregulates miR-34 expression and thus inhibits autophagy in HBV-expressing cells while HBx helps HBV evade DDX3-mediated inhibition by downregulating DDX3 expression in HBV-infected cells.     

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.     

体内与转染细胞中乙型肝炎病毒株复制特性的相关性

目的 比较不同乙型肝炎病毒（HBV）株在体内及转染细胞中复制特性是否相符。方法 以核酸杂交定量和聚合酶链反应-酶联免疫吸附试验（PCR-ELISA）测定5例孕妇血清中HBVDNA含量,并测定乙型肝炎表面抗原（HBsAg)和乙型肝炎e抗原（HBeAg)含量,分别克隆血清中的HBV基因组转染细胞,检测培养上清液中HBsAg和HBeAg表达水平,并以Southern印迹及核酸杂交检测转染细胞内、外HBVDNA复制水平。结果 转染细胞内、外HBVDNA复制水平与相应血清HBVDNA量呈正相关趋势,转染细胞表达的病毒抗原水平与相应血清中病毒抗原含量也呈正相关趋势。结论 感染者血清中HBVDNA和病毒抗原的含量与毒株在细胞中复制和抗原表达水平有相符趋势。HBV不同毒株在转染细胞中的复制可基本反映体内毒株的复制特性。     

Abnormal expression of HSP gp96 associated with HBV replication in human hepatocellular carcinoma

BACKGROUND: Heat shock protein (HSP) gp96 is a member of the HSP90 family and presumably overexpresses as a result of stimulation by mutated or abnormal proteins. Its abnormal expression correlates with carcinogenesis, progression and prognosis of hepatocellular carcinoma (HCC). In this study, we investigated the pathological characteristics of liver gp96 expression and its relationship with hepatitis B virus (HBV) replication in HCC patients. METHODS: Tumor specimens were prospectively collected from 30 HCC patients undergoing liver resection. Total RNAs were extracted from HCC or their noncancerous tissues. The distribution of gp96 expression in hepatocytes was investigated by streptavidin peroxidase (S-P) immunohistochemistry and tissue HBV-DNA was detected by the in situ molecular hybridization technique. The association of gp96 expression with HBV replication, and the histopathological characteristics of HCC were analyzed. RESULTS: The gp96 was strongly expressed in HCC (73.3%, 22 of 30) and weakly (46.7%, 14 of 30) in non-cancerous tissues. The gp96 expression in HCC tissues was correlated with degree of tumor differentiation and tumor size, but not with tumor number (P>0.05). Immunohistochemical analysis showed that 17 of 19 HCC patients with HBVDNA -positive were strongly expressed for gp96, whereas only 5 of 11 patients with HBV-DNA-negative were positive for gp96. A significant difference was found between the two groups (89.5% vs. 45.5%, P<0.05). CONCLUSIONS: The abnormal expressions of HSP gp96 in HCC tissues are associated with HBV replication. This finding indicates that HBV infection plays an important role in the development of HCC.     

靶向核糖核酸酶对乙型肝炎病毒复制的作用

通过定量检测上清液中的乙型肝炎e抗原(HBeAg)和乙型肝炎病毒脱氧核糖核酸(HBV DNA)，进一步研究靶向核糖核酸酶对HBV复制的影响。     

巨噬细胞外泌体抑制HBV DNA复制

目的探讨巨噬细胞(macrophage,M?)外泌体抑制HBV DNA复制的机制及与慢性乙型肝炎(chronic viral hepatitis B,CHB)肝损伤的相关性。方法体外分离鉴定M?外泌体,并将其与Hep G2.2.15细胞共培养,利用实时定量PCR检测共培养后HBV DNA复制的变化,分别分析M?及其上清外泌体中微小RNA(micro RNA,mi RNA)-638的表达;同时入组CHB免疫活化(immune activation,IA)期、低(非)复制(inactive carrier,IC)期患者,并以同期健康者作为对照组,通过实时定量PCR检测血清外泌体中mi RNA-638的表达,并与肝损伤、HBV DNA载量进行相关性分析。结果 M?外泌体可以抑制HBV DNA复制;M?及其上清外泌体中mi RNA-638高表达;CHB患者IA期mi RNA-638的表达水平低于IC期;CHB患者血清外泌体中mi RNA-638的表达水平与ALT、AST和HBV DNA水平呈负相关。结论 M?外泌体可以抑制HBV DNA复制,外泌体中mi RNA-638的表达水平与肝脏炎性损伤和病毒复制密切相关,M?外泌体相关mi RNA可能是潜在的抑制HBV复制、减轻肝脏炎症的靶点。     

慢性肝病患者乙型肝炎病毒BCP变异对HBV DNA复制水平影响的研究

目的探讨乙型肝炎病毒慢性感染中C基因启动子(BCP)变异对HBV DNA复制水平的影响及其临床意义。方法采用PCR微板核酸杂交结合ELISA检测显示技术,检测74例乙型肝炎病毒慢性感染者BCP区核苷酸(nt)1762碱基A→T和1764碱基G→A联合突变。结果在74例乙型肝炎病毒慢性感染者中检出BCP区T1762 A1764突变24例(32.4％),BCP变异阳性组的HBV DNA含量(10~(8.2992±0.8665)拷贝/ml)显著高于BCP变异阴性组的含量(10~(7.1737±1.1539)拷贝/ml ) P<0.001)。结论 BCP变异可引起HBV致病力增强,复制水平提高。     

C基因截短型HBV载体的复制、包装与表达

目的 探索C基因截短型重组HBV载体的复制、包装和表达。方法 采用分子克隆、定点突变技术构建C基因截短型HBV载体；瞬时转染HepG2细胞，在荧光显微镜下观察外源目的基因绿色荧光蛋白(GFP)的表达；提取转染细胞胞内、胞外DNA分别进行半巢式聚合酶链反应(PCR)、非变性凝胶电泳杂交技术、sourhern blot杂交定量分析及常规PCR等分析，检测重组HBV载体的复制、包装及子代病毒的生成。结果 经酶切鉴定，C基因截短型HBV载体构建成功；转染HepG2细胞，外源目的基因能够高效表达；在辅助载体的辅助下，C基因截短型HBV载体能够在肝细胞进行复制，包装，通过定量分析，C基因截短型HBV载体的包装效率比C基因非截短型HBV，载体提高4～8倍；另外，C基因截短型HBV载体可以在辅助载体的辅助下包装成携带外源目的基因的子代病毒颗粒分泌到胞外。结论 C基因部分截短不影响HBV载体外源基因的表达和病毒的复制，而且可以提高载体的包装效率。     

RNA干扰现象阻止丙型肝炎病毒基因表达和复制

1995年,康奈尔大学的Su Guo博士在用反义RNA阻断线虫基因表达的试验中发现,反义和正义RNA都能阻断基因的表达.1998年,Andrew的研究证明:在正义RNA阻断了基因表达的试验中,真正起作用的是双链RNA.这些小的双链RNA可以高效、特异的阻断体内特定基因表达,促使mRNA降解,诱使细胞表现出特定基因缺失的表型,称为RNA干扰(RNA interference,RNAi,也译作RNA干预或者干涉).它是体内抵御外在感染的一种重要保护机制.这些双链RNA是体外转录正义RNA时生成的.随后的研究中发现,RNAi现象广泛存在于植物、原生动物、昆虫、线虫和哺乳动物中[1].RNAi的最主要作用是可以特异性地使特定的基因沉默,从而起到控制细胞的各种高级生命活动.这为各种疾病如病毒病和肿瘤等的防治打开了一条全新的途径.由于RNAi的重要意义和近年来不断取得研究进展,<科学>杂志在2001年将其列为十大科学成就之一,2002年又将其列为当年十大科学成就之首.同时<自然>杂志也将小干扰RNA(small interfering RNAs,siRNA),评为2002年重大科技成果之一.     

rAAV载体介导的HDV核酶对HBV基因表达的抑制作用

目的探讨重组腺相关病毒介导的HDV核酶在细胞内抑制乙型肝炎病毒基因表达的作用。方法将针对HBV基因序列C区的反式HDVR z与U6启动子和绿色荧光蛋白基因EGFP及其启动子CMV共同插入腺相关病毒载体pSNAV中并取代其原有CMV启动子区域,构建为pSNAV-CR z重组载体。并将pSNAV-CR z、pSNAV及pLEGFP质粒转染HepG2.2.15培养细胞,荧光显微镜下观察绿色荧光,对转染后培养细胞内蛋白进行HBeAg和HBsAg的ELISA分析。结果所构建的重组载体经双酶切及PCR验证与实验设计一致。重组载体转染培养细胞后,荧光显微计数表明,pSNAV-CR z的转染效率为30%。HBeAg和HBsAg的ELISA检测显示,pSNAV-CR z重组载体对HepG2.2.15细胞中HBV病毒的HBeAg和HBsAg表达抑制率分别为63.5%和50.07%。结论细胞水平试验证明,重组腺相关病毒载体介导的反式HDVR z在体外培养细胞水平对HBV的复制起到一定的抑制作用。