初步探讨丙型肝炎病毒体外感染的致病机理

目的:初步探讨原代培养肝细胞对HCV的易感性,并在此基础上观察HCV感染后肝细胞形态学的变化。方法:用HCV RNA阳性血清与培养肝细胞共同孵育后,收集不同时相点标本,以逆转录聚合酶链反应(RT-PCR)、分别细胞内或上清中正负链RNA,免疫组化检测细胞内HCV NS3、NS5抗原的表达以及原位杂交检测细胞内HCV负链RNA,同时在电镜下观察细胞形态学变化。结果:接种感染血清后第3～5d,即可在细胞内或培养上清中检出HCV正负链RNA;感染肝细胞内可见HCV NS3、NS5抗原表达,阳性物质位于胞浆中;原位杂交法证实细胞内存在负链RNA,也位于胞浆中;光镜下未发现感染细胞形态学变化,电镜下发现胞浆内含有大量的脂肪空泡、多泡体,并可见疑似病毒样结构。结论:HCV不但能感染树鼩肝细胞,而且在体外能直接致细胞病变。     

EBV转化B淋巴细胞中丙型肝炎病毒抗原表达的免疫组化研究

观察丙型肝炎患者外周血单个核细胞（PBMC）体外转化培养后其中是否仍有丙型肝炎病毒（HVC）存在。用EB病毒感染患者HCV阳性PBMC7并使其转化,获得永生化B细胞（EBVTB）。而后,用套式RT-PCR检测培养细胞和上清中的HCV RNA,用免疫组化SP法观察HCV抗原在细胞中的表达,HCV在传代细胞中持续存在,而培养细胞中的HCVRNA负链和培养上清中的HCV间断阳性,免疫组化发现HCVNS3、NS5和C抗原分布于胞细胞膜和核膜周围也可见阳性着色,细胞核阴性。HCV可以在体外EB病毒转化B细胞系中较长时间存在和复制。     

多抗原肽免疫血清体外阻断丙型肝炎病毒感染外周血单个核细胞的研究

目的探讨多抗原肽（MAP）免疫兔血清体外阻断丙型肝炎病毒（HCV）感染人外周血单个核细胞。方法用兔抗MAP血清按一定比例混合后接种于HCV感染的人外周血单个核细胞,采用逆转录聚合酶链反应、免疫组化和原位杂交技术分别检测细胞内或上清中HCV RNA、细胞内HCV NS3和HCV NS5抗原的表达。结果兔抗MAP血清按1：1混合后,外周血单个核细胞仍可检出HCV RNA,细胞有HCV NS3抗原表达;而按5：1和10：1混合后,单个核细胞内未检测出HCV正负链RNA,未见特异性抗原表达,原位杂交也未检测出HCV RNA存在。结论兔抗MAP血清在体外能部分阻断HCV感染。     

体外感染实验探讨丙型肝炎病毒的ADE分子机制

目的研究丙型肝炎病毒(HCV)在感染人滋养层细胞的过程中是否存在抗体依赖性感染增强(ADE)作用,以探讨HCV母婴传播的分子机制。方法将HCV阳性血清以4种不同方式感染人滋养层细胞,以免疫电镜观察HCV病毒颗粒在滋养层细胞中的表达,应用RT-PCR法、免疫组化法检测细胞内外的HCVRNA正链、负链,HCVNS5、NS3及C区抗原。结果在滋养层细胞胞浆内发现了HCV病毒颗粒,HCVRNA正链、负链,HCVNS5、NS3、C区抗原仅在全血清感染细胞内或上清中间断测得。结论HCV可以在滋养层细胞中复制。HCV感染滋养层细胞的过程中存在ADE机制,抗体和补体均参与了HCV的跨膜转运过程。     

丙型肝炎病毒体外感染胎肝细胞的初步研究

目的：探索原代培养胎肝细胞对丙型肝炎病毒（HCV）的易感性,旨在建立较为稳定实用的细胞感染模型。方法：研究血清与培养肝细胞共同孵育6－8h后,收集不同时相点标本,用逆转录聚合酶链反应（RT－PCR）分别检测细胞内或上清液中正负链RNA,免疫组化检测细胞内HCV NS3,NS5特异性抗原的表达情况,以及原位杂交检测细胞内HCV负链RNA。结果：接种感染血清3d后,即可在细胞内或培养上清液中检出HCV正负链RNA,间断检出至感染后第17天,HCV NS3,NS5特异性抗原可在感染细胞内表达,阳性物质位于乐中,原位杂交法证实细胞内存在负链RNA,也位于胞浆中,结论：原代培养的胎肝细胞对HCV不但易感,而且稳定地支持HCV复制。     

肝病患者肝组织HCV抗原的检测及意义

目的 探讨肝病患者肝组织丙型肝炎病毒（HCV）抗原的表达及意义。方法 应用免疫组织化学方法对252例肝病患者石蜡包埋肝组织中HVC抗原进行了检测。结果 多克隆抗HCV检出HcAg阳性25例（9．92％），单克隆抗HCV-NS3检出HCV—NS3蛋白阳性19例（7．54％）。19例HCV—NS3阳性肝组织均同时表现HcAg阳性，二者在肝细胞分布状况相似，不同病理类型肝炎患者肝组织中HCV抗原检出率无统计学意义。252例血清检出HCV标志物阳性71例，单独抗HCV阳性者无1例肝组织中检出抗原。结论 肝组织中抗原的检出和血清HCV标志具有良好对应性，但不同血清HCV标志模式抗原检出率有统计学意义。     

丙型肝炎病毒体外感染肝癌细胞株HepG2的初步研究

目的 研究人肝癌细胞株HepG2对丙型肝炎病毒（HCV）的易感性，建立细胞感染模型。方法 将人肝癌细胞株HepG2与慢性丙型肝炎患者血清共同温育6-8h，收集不同时相点标本，用逆转录聚合酶链反应（RT-PCR)法分别检测细胞内或上清液中正负链RNA，免疫组织化学法检测细胞内HCV NS3，NS5特异性抗原的表达情况，以及原位杂交法检测细胞内HCV负链RNA。结果 接种感染血清3-35d，可在细胞内或培养上清液中检出HCV正负链RNA；HCVNS3，NS5特异性抗原能在感染细胞内稳定表达，阳性物质位于胞浆中；原位杂交法证实细胞内存在负链RNA，也位于胞浆中，结论 人肝癌细胞株HepG2不但对HCV易感，而且可以稳定地支持HCV体外复制。     

HepG2细胞中HCV NS5A蛋白对HCV IRES启动蛋白翻译的影响

目的探讨HepG2细胞中HCV非结构蛋白5A（NS5A）对HCV IRES启动蛋白翻译的影响，以了解HCV的复制调控机制。方法将构建的表达双荧光素酶的双顺反子载体pCMV-Rluc-IRES-Fluc和含HCV NS5A基因的表达质粒pcDNA-NS5A共转染HepG2细胞，用双荧光素酶检测系统检测虫荧光素酶的表达水平，细胞免疫荧光技术检测HCV-NS5A蛋白的表达，RT-PCR检测虫荧光素酶基因mRNA水平，并与相应对照做比较，以观察HCV NS5A对HCV IRES介导虫荧光素酶翻译水平的影响。结果转染pcDNA-NS5A的HepG2细胞中虫荧光素酶活性明显高于转染pCDNA3．I-3flag的对照组，并存在剂量依赖关系；而RT-PCR虫荧光素酶基因mRNA水平在两组间差异无统计学意义。转染pcDNA-NS5A的HepG2细胞质中可见HCV NS5A蛋白的表达。结论HCV NS5A蛋白对HCV IRES介导虫荧光素酶的翻译有正调节作用，并存在剂量依赖关系．     

丙型肝炎病毒非结构蛋白4B对肝癌细胞增殖的影响

目的 探讨丙型肝炎病毒非结构蛋白4B(HCV-NS4B)对肝癌细胞增殖及增殖细胞核抗原(PCNA)和周期素D1(cyclinD1)蛋白表达的影响.方法 采用构建好的HCV-NS4B表达载体,以脂质体转染法转染HepG2细胞.RT-PCR、琼脂糖凝胶电泳证实HCV-NS4B在HepG2细胞稳定表达.MTT法绘制生长曲线,观察NS4B对肝细胞生长的影响;流式细胞仪检测细胞周期的情况;免疫细胞化学法检测PCNA和cyclinD1的表达.结果 转染后,生长曲线显示NS4B可促进肝细胞生长;细胞周期检测显示转染NS4B的HepG2细胞进入S期和G2/M期增多(P＜0.05),处于G0/G1期细胞降低(P＜0.05).PCNA和cyclinD1的表达均较空白载体组升高(P＜0.05).结论 HCV-NS4B可干扰肝癌细胞周期,促进肝癌细胞增殖,其作用与上调PCNA、cyclinD1表达有关.     

激光共聚焦技术证实HCV NS5在人胎盘滋养层细胞的定位表达

目的胎盘屏障是营养物质以及某些药物、病原体、激素等从母体进入胎儿的必经之路。体外分离培养滋养层细胞是研究其功能及母体与胎儿之间物质交换的具体分子机制的细胞学基础。本研究的目的是探讨体外分离培养的人体胎盘滋养层细胞感染丙型肝炎病毒(HCV)后HCV NS5在滋养层细胞中的定位表达。方法采用胰蛋白酶消化法及Percoll密度梯度分离法分离培养人胎盘组织中滋养层细胞,以HCV RNA阳性血清对滋养层细胞进行体外感染试验,应用RT-PCR法定性及定量检测感染后细胞培养上清中HCV RNA,用激光共聚焦技术观察免疫荧光染色后HCV NS5在滋养层细胞中的定位。结果HCV感染后的细胞培养上清中可间断检测到HCV RNA,而对照组始终未检出;激光共聚焦观察显示HCV NS5主要定位于细胞核周围。结论感染HCV的人滋养层细胞中存在HCV NS5表达。此研究为进一步深入研究HCV的母婴传播分子机制奠定了实验基础。     

Effect of fragile histidine triad gene transduction on proliferation and apoptosis of human hepatocellular carcinoma cells

AIM： To evaluate the inhibitory effects of human fragile histidine triad （FHIT） gene on cell proliferation and apoptosis in human hepatocellular carcinoma line Hep3B in vitro.
METHODS： A recombinant pcDNA3.1 （＋）/FHIT including the functional region of FHIT gene was constructed and transferred into human hepatocellular carcinoma cells in vitro, mRNA and protein expression of the FHIT gene in the transfected cells was detected by RT-PCR and Western blot, respectively. The effect of FHIT on proliferation was detected by MTT assay. Changes in cell cycle and apoptosis were assayed by flow cytometry. Five mice received subcutaneous transplantation of Hep3B-FHIT; 5 mice received subcutaneous transplantation of normal Hep3B and Hep3B-C as controls. The body weight of nude mice and tumor growth were measured.
RESULTS： RT-PCR and Western blot analysis showed that the expression level of FHIT-mRNA and FHIT protein was higher in Hep3B cells after infection with pcDNA3.1 （＋）/FHIT. The growth of Hep3B cells treated with pcDNA3.1 （＋）/FHIT was significantly inhibited. The pcDNA3.1 （＋）/FHIT-transfected Hep3B cells showed a significantly higher cell rate at G0-G1 phase and increased apoptosis in comparison with controls （P 〈 0.05）. The growth of transplanted tumor was inhibited markedly by FHIT. Tumors arising from the Hep3B-FHIT cells occurred much later than those arising from the Hep3B and Hep3B-C cells. The growth of Hep3B-FHIT cells was slow and the tumor volume was low.
CONCLUSION： Transduction of FHIT gene inhibits the growth of human hepatocellular carcinoma cells and induces cell apoptosis in vivo and in vitro.     

Effects of Ginkgo biloba extract on cell proliferation and cytotoxicity in human hepatocellular carcinoma cells

AIM: To study the effect of Ginkgo biloba extract (EGb 761) containing 22-27% flavonoids (ginkgo-flavone glycosides) and 5-7% terpenoids (ginkgolides and bilobalides) on cell proliferation and cytotoxicity in human hepatocellular carcinoma (HCC) cells. METHODS: Human HCC cell lines (HepG2 and Hep3B) were incubated with various concentrations (0-1 000 mg/L) of EGb 761 solution. After 24 h incubation, cell proliferation and cytotoxicity were determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and lactate dehydrogenase (LDH) release, respectively. After 48 h incubation, the expression of proliferating cell nuclear antigen (PCNA) and p53 protein was measured by Western blotting. RESULTS: The results showed that EGb 761 (50-1 000 mg/L) significantly suppressed cell proliferation and increased LDH release (P<0.05) in HepG2 and Hep3B cells compared with the control group. The cell proliferation of HepG2 and Hep3B cells treated with EGb 761 (1 000 mg/L) was 45% and 39% of the control group (P<0.05), respectively. LDH release of HepG2 cells without and with EGb 761 (1 000 mg/L) treatment was 6.7% and 37.7%, respectively, and that of Hep3B cells without and with EGb 761 (1 000 mg/L) treatment was 7.2% and 40.3%, respectively. The expression of PCNA and p53 protein in HepG2 cells treated with EGb 761 (1 000 mg/L) was 85% and 174% of the control group, respectively. CONCLUSION: Ginkgo biloba extract significantly can suppress proliferation and increase cytotoxicity in HepG2 and Hep3B cells. Additionally, Ginkgo biloba extract can decrease PCNA and increase p53 expression in HepG2 cells.     

人肝癌细胞系7721丙型肝炎病毒体外感染模型的建立

目的　建立接近体内自然感染状态并能稳定支持丙型肝炎病毒（ＨＣＶ） 体外长期复制的感染细胞模型。方法　将人肝癌细胞系７７２１ 与慢性丙型肝炎患者血清共温育８ 小时后，分别以逆转录聚合酶链反应、免疫组化、原位杂交检测细胞和／ 或培养上清中的ＨＣＶ ＲＮＡ、ＨＣＶ 抗原表达及ＨＣＶＲＮＡ 在感染细胞内的定位。结果　感染血清和细胞共同温育后的第２ ～６６ 天，从细胞内和培养上清中均可间断地检测出ＨＣＶ 正、负链ＲＮＡ，即使其间细胞传代６ 次，ＨＣＶ ＮＳ３ 、ＮＳ５ 抗原在细胞内能稳定表达，原位杂交显示ＨＣＶ ＲＮＡ 阳性物质多位于细胞浆。结论　７７２１ 细胞不但对ＨＣＶ 易感，而且可以稳定地支持ＨＣＶ 的体外长期复制，此模型可以用于ＨＣＶ 感染、复制机制的研究、抗病毒药物的评价及保护性抗体／ 疫苗的初步筛选。     

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

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特异性寡聚脱氧核苷酸对Hep3B肝癌细胞恶性表型的作用

目的研究一段序列和胰岛素样生长因子2(IGF-2)基因第四启动子互补的特异性寡聚脱氧核苷酸(SODN)对Hep3B肝癌细胞株的抑制效应。方法根据IGF-2基因第四启动子序列人工合成一段与之互补的特异性脱氧核苷酸,并制备其长效的硫代衍生物,通过转染试剂将其导入高表达IGF-2的Hep3B 肝癌细胞株后,采用逆转录聚合酶链反应法和western blot技术分析SODN对IGF-2基因转录和蛋白表达的抑制效应,并通过四甲基偶氮唑盐法、流式细胞术、细胞克隆形成实验以及细胞侵袭和运动实验进一步观察SODN对Hep3B细胞的生长、细胞周期、体外克隆形成能力及侵袭和运动能力所产生的影响。结果空白对照组和导入非特异性寡聚脱氧核苷酸(CODN)对照组相比,导入SODN的Hep3B细胞,其IGF-2 mRNA 和蛋白的表达有明显降低;SODN对Hep3B细胞的增殖及运动能力不产生效应,但可以抑制其在培养板上的克隆形成数和穿透Matrigel的细胞数,抑制率分别为90.2%、95.5%。结论SODN具有"分子开关"作用,可以抑制和下调Hep3B肝癌细胞的IGF-2基因表达,并可逆转其部分恶性表型。     

Overexpression of the mouse Fas gene in human Hep3B hepatoma cells overcomes their resistance to Fas-mediated apoptosis

BACKGROUND/AIMS: Fas-induced apoptosis is one of the main forms of apoptosis occurring in hepatocytes. We have previously demonstrated that the human hepatoma cell line Hep3B is resistant to Fas-mediated apoptosis. In this study, we investigated whether the human Fas receptor itself, or the Fas transduction pathway was responsible for the resistant phenotype. METHODS: Clones of Hep3B cells overexpressing the mouse Fas gene (Hep3B(mfas)) were generated by transfection, and apoptosis was studied by (i) chromatin condensation and nuclear fragmentation, (ii) flow cytometry, (iii) DNA fragmentation and (iv) poly (ADP-ribose) polymerase cleavage. RESULTS: Use of the species-specific and agonistic anti-mFas monoclonal antibody (JO2), showed that the mFas receptor was correctly routed to the plasma membrane of Hep3B(mfas) cells. Using the four above-mentioned criteria, we demonstrated that JO2 triggered mFas-mediated apoptosis of Hep3B(mfas), but not of Hep3B(pCi) cells (transfected with an empty vector). CONCLUSIONS: Our data show (i) that the Fas signaling pathway can be completed when a functional mFas receptor is expressed in Hep3B cells, and thus, (ii) that the death-inducing signaling complex components and the effector caspases are functional in Hep3B cells. Moreover, they suggest that the Fas subunits are not pre-assembled at the cell membrane before receptor-ligand interaction.     

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

AIM: To establish a cell culture system with longterm replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro. METHODS: HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively. RESULTS: The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION: HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.