高复制HBV转基因小鼠模型对抗病毒药物拉米夫定评价的研究

[目的]探讨乙型肝炎病毒(HBV)转基因小鼠模型筛选抗HBV药物的可行性。[方法]用公认抗HBV复制药物拉米夫定对我们建立高复制HBV转基因鼠进行实验,选我们建立的1.3copy高复制HBV转基因小鼠20只,随机分成两组,每组10只。采用灌胃针灌胃法给药。对照组灌喂生理盐水,实验组灌喂拉米夫定,剂量为100mg/kg,每天2次,连续灌21d,每7d采血1次。荧光定量PCR检测血清中HBVDNA。[结果]实验组用拉米夫定前小鼠血清HBVDNA5.50±0.42(拷贝数log10数值),3周后HBVDNA已显著降低(4.63±0.57),4周后,小鼠血清HBVNDA为4.08±0.51,停药1周后,再次检测血清HBVDNA,小鼠血清HBVDNA又恢复正常水平(5.70±0.39)。[结论]我们建立的高复制HBV转基因小鼠模型验证了拉米夫定对HBV复制的抑制程度和持续时间,表明该模型可应用于抗HBV药物的筛选、评价研究。     

应用慢病毒载体携带HBV基因组DNA制备乙型肝炎病毒感染小鼠模型的影响因素分析

应用慢病毒载体构建不同HBV转导质粒,通过高压水动力法尾静脉注射小鼠,比较不同HBV转导质粒、剂量(5μg和10μg)、小鼠品系(Balb/c和C57BL/6)及鼠龄(6周龄和18周龄)对建立HBV感染模型的影响。不同的时间点尾静脉采血,ELISA检测血清HBsAg、HBeAg的表达水平及动力变化,Real-time PCR检测血清及肝组织病毒载量;免疫组织化学法检测肝组织HBcAg的定位与表达。1.3倍HBV基因组慢病毒载体转导质粒(pCSHBV1.3)优于1.1倍与1.2倍HBV基因组转导质粒(pCS-HBV1.1or pCS-HBV1.2);pCS-HBV1.3注射Balb/c小鼠后抗原表达维持时间短,抗体出现早;pCS-HBV1.3注射C57BL/6小鼠后,HBsAg、HBeAg抗原表达及血清HBV DNA水平维持时间长;且注射5μg质粒相对于10μg质粒注射小鼠后抗原表达维持时间更长;而6周龄和18周龄小鼠血清均可在较长时间内检测到HBsAg、HBeAg及HBV DNA的表达,但在注射后35周内,前者的表达量均高于后者;所有注射质粒的小鼠肝组织中均可检测到HBcAg的表达,且在血清HBV感染标志转阴时均可检测到肝内HBV DNA的存在。注射质粒的HBV基因组长度、剂量以及宿主的遗传背景均对建立乙肝成体转基因小鼠模型有影响,且发现以5μg含1.3倍HBV基因组的转导质粒pCS-HBV1.3注射6周龄C57BL/6小鼠,HBV抗原表达和HBV DNA水平维持时间长,更适合建立HBV持续感染模型。     

用rAAV8-1.3HBV制备两种品系小鼠乙型肝炎病毒感染模型的比较研究

我们先前用rAAV8-1.3HBV静脉注射C57BL/6小鼠成功地制备了慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染模型。为了探讨不同品系的小鼠对rAAV8-1.3HBV静脉注射是否具有不同反应,本研究比较了C57BL/6和BALB/c小鼠静脉注射重组病毒后外周血中HBV抗原和抗体水平、病毒载量和肝脏组织HBcAg表达情况,以及不同剂量重组病毒注射与这些指标的关系。将低(4×109 Viral genome,vg)、中(4×1010vg)和高(4×1011vg)三种剂量的rAAV8-1.3HBV通过尾静脉注射至C57BL/6和BALB/c小鼠,分别利用ELISA和荧光定量PCR方法检测血清中的HBV抗原、抗体水平以及HBV DNA,利用免疫组化检测肝脏组织HBcAg的表达。结果发现,对于C57BL/6小鼠,三种不同剂量rAAV8-1.3HBV注射均可造成100%小鼠出现HBV持续感染;血清HBsAg、HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,其表达水平随重组病毒注射剂量的增加而升高,高剂量注射时可造成超过40%的肝细胞感染HBV,血清中HBV DNA可达105 IU/mL以上;未检测到针对HBV的抗体。对于BALB/c小鼠,三种不同剂量rAAV8-1.3HBV注射也可造成100%小鼠出现HBV持续感染;血清HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,但是血清HBsAg在重组病毒注射2周之后显著下降甚至消失;在中剂量注射组的BALB/c小鼠血清中检测到低水平的Anti-HBs;血清HBeAg和肝组织HBcAg的表达水平随重组病毒注射剂量的增加而增高,并且各剂量组表达水平均高于C57BL/6小鼠,高剂量注射时可造成超过50%的肝细胞感染HBV。本研究表明,低至4×109 vg剂量的rAAV8-1.3HBV注射即可造成C57BL/6和BALB/c两种品系小鼠出现HBV持续感染,并且HBV复制水平随重组病毒注射剂量增加而增高;BALB/c小鼠对HBV的免疫反应强于C57BL/6小鼠,可以产生针对HBsAg的体液免疫反应而使血清HBsAg转阴,但无法清除携带HBV的肝细胞。     

C57BL/6J-HBV转基因小鼠随增龄乙肝表面抗原表达及转氨酶水平变化探讨

目的 探讨C57BL/6J-HBV乙肝转基因小鼠随增龄表面抗原表达及转氨酶水平的变化.方法 经血清表面抗原检测,根据S/C.O值将乙肝转基因小鼠分为两组,两组小鼠在4～10周龄每隔一周以酶联免疫吸附法测定血清表面抗原水平;各组小鼠在5～11周龄每隔一周以全自动生化分析仪测定小鼠血清谷丙转氨酶(ALT)和谷草转氨酶(AST)水平.结果 实验1组HBV乙肝转基因小鼠表面抗原表达6周龄与4周龄相比显著增高,6周龄至10周龄趋于稳定;实验2组的雄性小鼠表面抗原表达6周龄与4周龄相比极显著降低(P＜0.01),而雌性小鼠在4周龄至10周龄表面抗原表达相对稳定.实验1组的雄性小鼠ALT 水平9周龄与7周龄比较显著下降(P＜0.05),其余小鼠在5周龄至11周龄呈现下降趋势,但无显著性差异.实验1组的雄性小鼠AST水平11周龄与7周龄相比显著升高(P＜0.05).结论 乙肝表面抗原S/C.O值不同的转基因小鼠表面抗原表达随增龄变化趋势不同;血清谷丙转氨酶和谷草转氨酶水平在5～11周龄变化趋势相反.     

乙型肝炎病毒急性感染小鼠模型的建立

采用高压水注射方法,通过尾静脉将具有复制能力的HBV质粒导入BABL/cJ小鼠体内,应用real-time PCR、ELISA、RIA、Southern Blot、Northern Blot,以及免疫组化等方法,检测小鼠病毒血症、血清和肝组织中HBV 抗原表达动态变化、肝组织中HBV转录和复制情况,以及小鼠免疫应答状况。结果HBV基因可以在小鼠体内表 达和复制,并诱导小鼠产生特异性免疫应答,其应答模式及HBV清除过程与人类的HBV急性感染类似。实验显 示高压注射具有复制能力的HBV质粒可以在小鼠体内建立HBV急性感染模型,这种模型可以用于HBV病毒 学、免疫学以及抗病毒药物筛选等方向的研究。     

丙型肝炎病毒NS3-5B转基因小鼠模型的构建

目的:构建表达丙型肝炎病毒(HCV)NS3-5B蛋白的转基因小鼠模型。方法:显微注射线性化p IRES2-EGFP-NS3-5B载体到小鼠受精卵,制备并传代筛选HCV NS3-5B转基因小鼠;通过血清谷丙转氨酶(ALT)、谷草转氨酶(AST)检测和肝脏HE染色,对6周龄转基因小鼠进行肝功能评价。结果:PCR、RT-PCR和Western印迹结果表明HCV NS3-5B转基因小鼠构建成功;6周龄部分转基因小鼠血清ALT和AST值升高,但差别无统计学意义,肝组织形态无改变。结论:构建了HCV NS3-5B转基因小鼠模型,为进一步在体研究HCV复制复合体建立了平台。     

比较不同血清型AAV携带HBV基因组建立乙肝小鼠模型效果

近年来,用8型腺相关病毒携带1.3拷贝HBV(Hepatitis B virus)基因组建立的HBV持续感染小鼠模型受到越来越多的关注。本研究比较了除AAV8之外的其他4种血清型重组腺相关病毒(Recombinant adeno-associated virus,rAAV)建立乙肝小鼠模型效果。首先,将携带1.3拷贝ayw亚型HBV基因组的1型、2型、5型、8型、9型腺相关病毒分别以1×10~(11) vg/只(Viral genome,vg)的剂量尾静脉注射C57BL/6J小鼠;利用ELISA方法监测小鼠血清中HBeAg和HBsAg表达水平;用定量PCR方法检测小鼠血清和肝脏中HBV DNA拷贝数;用免疫组化方法检测小鼠肝脏中HBc Ag的表达;用HE染色检测小鼠肝脏病理变化。结果显示,在持续8周中,5组小鼠血清中都检测到HBeAg和HBsAg的表达,血清和肝脏中均检测到HBV DNA的存在。HBeAg、HBsAg、HBV DNA表达水平高低依次为AAV8AAV9AAV1AAV5AAV2。5组小鼠用免疫组化方法都检测到肝脏中HBcAg表达,HE染色病理检测均观察到不同程度的肝损伤。本研究扩大了能用于建立乙肝小鼠持续感染模型可选择的AAV载体种类,发现虽然AAV1、2、5、9的建模效果不如AAV8,但它们都可以介导建立持续感染的乙肝小鼠模型,建模效果依次为AAV8AAV9AAV1AAV5AAV2。其中AAV9介导的建模效果与AAV8载体最为接近,可以替代AAV8载体用于有效地建立HBV持续感染的小鼠模型。     

NIRF可抑制乙型肝炎病毒在水动力法转染HBV小鼠模型中的复制及表达

目前对NIRF(Np95/ICBP-90 like RING finger protein)的研究正朝着细胞癌变进程以及表观遗传学的方向发展,但在体内NIRF对乙型肝炎病毒(HBV)的复制及表达的影响,目前尚不明确.通过高压水动力法转染HBV小鼠模型,不同时间点收集血液和肝组织标本,荧光定量PCR检测血清及肝组织中病毒载量,WesternBlot检测肝组织HBc(hepatitis B virus core protein)表达,ELISA检测血清HBeAg表达,并通过免疫组化染色检测HBsAg、HBcAg在肝组织中的定位及表达.小鼠转染pAAV-HBV1.3和NIRF以后,血清及肝组织病毒载量降低(n=3,P<0.05),HBc蛋白及HBV相关抗原的表达受到抑制,说明在水动力法转染HBV小鼠模型中NIRF对HBV的复制及表达起到抑制作用,期待能为后续的HBV致病机理及治疗药物的研究与开发提供支持与帮助.     

乙型肝炎病毒急性感染小鼠模型的建立

采用高压水注射方法,通过尾静脉将具有复制能力的HBV质粒导入BABL/cJ小鼠体内,应用real-timePCR、ELISA、RIA、Southern Blot、Northern Blot,以及免疫组化等方法,检测小鼠病毒血症、血清和肝组织中HBV抗原表达动态变化、肝组织中HBV转录和复制情况,以及小鼠免疫应答状况.结果HBV基因可以在小鼠体内表达和复制,并诱导小鼠产生特异性免疫应答,其应答模式及HBV清除过程与人类的HBV急性感染类似.实验显示高压注射具有复制能力的HBV质粒可以在小鼠体内建立HBV急性感染模型,这种模型可以用于HBV病毒学、免疫学以及抗病毒药物筛选等方面的研究.     

c—Ha-ras在转基因小鼠模型C57-ras各组织中的表达谱分析

目的：分析转基因c-Ha-ras在C57-ras癌症小鼠模型中的组织表达谱及时空表达差异。方法：利用半定量及荧光定量RT-PCR法,分析不同首建鼠系、不同周龄小鼠各脏器中转基因c-Ha-ras的表达。结果：转基因c-Ha-ras在心、肝等13种组织器官中均有表达,在肺脏中表达最高,在肝脏中表达最低,No.2、No.3和No.5等3个首建鼠均呈现相似的变化规律;转基因在No.5首建鼠中表达水平最高,而在同一个首建鼠系中,12周龄时表达高于8周龄和24周龄。结论：转基因c-Ha-ras在各脏器中能高效表达,并间接表明该转基因能稳定遗传,为C57-ras癌症小鼠模型用于新药临床前致癌性评价提了供理论支持。     

siRNA Combinations Mediate Greater Suppression of Hepatitis B virus Replication in Mice

Hepatitis B virus (HBV) infection is a major world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome shortcomings of established drugs. In this study, the inhibitory effect mediated by combination of siRNAs targeting different sites of HBV in transgenic mice was analyzed. HBsAg and HBeAg in the sera of the mice were analyzed by enzyme-linked immunoadsorbent assay, HBV DNA by real-time PCR and HBV mRNA by RT-PCR. Our data demonstrated that all the three siRNAs employed showed marked anti-HBV effects. The expression of HBsAg and the replication of HBV DNA could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, combination of siRNAs compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA used for therapy was the same. Secreted HBsAg and HBeAg in the serum of mice treated with siRNA combination were reduced by 96.7 and 96.6 %, respectively. Immunohistochemical detection of liver tissue revealed 91 % reduction of HBsAg-positive cells in the combination therapy group. The combination of siRNAs caused a greater inhibition in the levels of viral mRNA and DNA (90 and 87.7 %) relative to the control group. It was noted that the siRNA3 showed stronger inhibition of cccDNA (78.6 %). Our results revealed that combination of siRNAs mediated a stronger inhibition of viral replication and antigen expression in transgenic mice than single siRNAs.     

大剂量HBsAg对HBV转基因小鼠T细胞免疫调节的研究

目的：研究大剂量HBsAg对HBV转基因小鼠其T细胞免疫效果的影响。方法：用大剂量血源性HBsAg免疫HBV转基因小鼠,采用ELISA方法观察转基因小鼠所诱生的HBsAg特异性Th1类细胞因子的水平,ELISPOT方法检测不同免疫方案对小鼠HBsAg特异性分泌IFN-γT细胞数量的影响,同时检测对小鼠淋巴细胞增殖的影响。结果：HBsAg组免疫后脾细胞产生的Th1类细胞因子（IFN-γ、IL-2）、HBsAg特异性分泌IFN-γT细胞及T细胞增殖水平较对照组显著增加（P〈0.05）。结论：大剂量的HBs-Ag可以诱导乙肝转基因小鼠产生高水平Th1类细胞因子并打破免疫耐受。     

Persistence of the Recombinant Genomes of Woodchuck Hepatitis Virus in the Mouse Model

Hydrodynamic injection (HI) with a replication competent hepatitis B virus (HBV) genome may lead to transient or prolonged HBV replication in mice. However, the prolonged HBV persistence after HI depends on the specific backbone of the vector carrying HBV genome and the genetic background of the mouse strain. We asked whether a genetically closely related hepadnavirus, woodchuck hepatitis virus (WHV), may maintain the gene expression and replication in the mouse liver after HI. Interestingly, we found that HI of pBS-WHV1.3 containing a 1.3 fold overlength WHV genome in BALB/c mouse led to the long presence of WHV DNA and WHV proteins expression in the mouse liver. Thus, we asked whether WHV genome carrying foreign DNA sequences could maintain the long term gene expression and persistence. For this purpose, the coding region of HBV surface antigen (HBsAg) was inserted into the WHV genome to replace the corresponding region. Three recombinant WHV-HBV genomes were constructed with the replacement with HBsAg a-determinant, major HBsAg, and middle HBsAg. Serum HBsAg, viral DNA, hepatic WHV protein expression, and viral replication intermediates were detected in mice after HI with recombinant genomes. Similarly, the recombinant genomes could persist for a prolonged period of time up to 45 weeks in mice. WHV and recombinant WHV-HBV genomes did not trigger effective antibody and T-cell responses to viral proteins. The ability of recombinant WHV constructs to persist in mice is an interesting aspect for the future investigation and may be explored for in vivo gene transfer.     

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。     

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.     

Inhibition of the HCV Core Protein on the Immune Response to HBV Surface Antigen and on HBV Gene Expression and Replication In Vivo

The hepatitis C virus (HCV) core protein is a multifunctional protein that can interfere with the induction of an immune response. It has been reported that the HCV core protein inhibits HBV replication in vitro. In this study, we test the effect of the HCV core gene on the priming of the immune response to hepatitis B surface antigen (HBsAg) and on the replication of HBV in vivo. Our results showed that the full-length HCV core gene inhibits the induction of an immune response to the heterogeneous antigen, HBsAg, at the site of inoculation when HCV core (pC191) and HBsAg (pHBsAg) expression plasmids are co-administered as DNA vaccines into BALB/c mice. The observed interference effect of the HCV core occurs in the priming stage and is limited to the DNA form of the HBsAg antigen, but not to the protein form. The HCV core reduces the protective effect of the HBsAg when the HBsAg and the HCV core are co-administered as vaccines in an HBV hydrodynamic mouse model because the HCV core induces immune tolerance to the heterogeneous HBsAg DNA antigen. These results suggest that HCV core may play an important role in viral persistence by the attenuation of host immune responses to different antigens. We further tested whether the HCV core interfered with the priming of the immune response in hepatocytes via the hydrodynamic co-injection of an HBV replication-competent plasmid and an HCV core plasmid. The HCV core inhibited HBV replication and antigen expression in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, the mouse models of acute and chronic hepatitis B virus infections. Thus, the HCV core inhibits the induction of a specific immune response to an HBsAg DNA vaccine. However, HCV C also interferes with HBV gene expression and replication in vivo, as observed in patients with coinfection.     

Hepatitis B Virus-Specific miRNAs and Argonaute2 Play a Role in the Viral Life Cycle

Disease-specific serum miRNA profiles may serve as biomarkers and might reveal potential new avenues for therapy. An HBV-specific serum miRNA profile associated with HBV surface antigen (HBsAg) particles has recently been reported, and AGO2 and miRNAs have been shown to be stably associated with HBsAg in serum. We identified HBV-associated serum miRNAs using the Toray 3D array system in 10 healthy controls and 10 patients with chronic hepatitis B virus (HBV) infection. 19 selected miRNAs were then measured by quantitative RT-PCR in 248 chronic HBV patients and 22 healthy controls. MiRNA expression in serum versus liver tissue was also compared using biopsy samples. To examine the role of AGO2 during the HBV life cycle, we analyzed intracellular co-localization of AGO2 and HBV core (HBcAg) and surface (HBsAg) antigens using immunocytochemistry and proximity ligation assays in stably transfected HepG2 cells. The effect of AGO2 ablation on viral replication was assessed using siRNA. Several miRNAs, including miR-122, miR-22, and miR-99a, were up-regulated at least 1.5 fold (P<2E-08) in serum of HBV-infected patients. AGO2 and HBcAg were found to physically interact and co-localize in the ER and other subcellular compartments. HBs was also found to co-localize with AGO2 and was detected in multiple subcellular compartments. Conversely, HBx localized non-specifically in the nucleus and cytoplasm, and no interaction between AGO2 and HBx was detected. SiRNA ablation of AGO2 suppressed production of HBV DNA and HBs antigen in the supernatant.

Conclusion

These results suggest that AGO2 and HBV-specific miRNAs might play a role in the HBV life cycle.