丙型肝炎病毒对SMMC 7721细胞体外直接致病作用的研究

目的:探讨丙型肝炎病毒(HCV)的直接致病作用。方法:用HCV阳性血清体外感染SMMC 7721细胞,观察细胞形态的变化,并运用四甲基偶氮唑盐(MTT)法及流式细胞仪(FCM)分别检测培养细胞的存活率及凋亡率,同时用套式逆转录-聚合酶链反应(nest RT-PCR)检测其中的正、负链HCV RNA。结果:HCV感染后部分SMMC7721细胞发生皱缩、脱壁,8h细胞存活率为74.2％,细胞凋亡率为18.7％,至56h后细胞存活率回复至84.3％以上,而细胞凋亡率又降至7.4％以下;各时间组的细胞存活率显著低于未感染HCV的SMMC7721细胞,而凋亡率则显著高于未感染的SMMC 7721细胞。感染后24h细胞及上清中HCV RAN检测结果均阴性,48h后在细胞中可检出正、负链HCV RNA,96h后细胞中HCV RNA正链呈阳性、负链转为阴性。结论:1.HCV具有一定的直接致病作用;2.HCV感染SMMC7721后的复制不稳定。     

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。     

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。     

丙型肝炎病毒体外可感染树鼩肝细胞

目的 探讨丙型肝炎病毒（HCV）体外感染树目句原代肝细胞。方法 用HCV RNA阳性血清感染原代树鼩肝细胞，并用感染后细胞培养上清液进行传代感染，通过检测受染肝细胞正、负链HCV RNA、培养上清液中包装后HCV RNA，并对比分析感染前后病毒准种变化等指标，评价感染是否成功。结果 受染树鼩肝细胞自第5～10天可检出负链HCV RNA，而正链RNA至感染后第14天仍可检出；感染后3～14d培养上清液中可检出HCV RNA，且呈RNA酶抗性；培养上清液中病毒可传代感染新的树鼩肝细胞；感染前后HCV准种分析显示树鼩肝细胞可被特定的准种选择性感染，而传代感染后肝细胞可检出新的准种。结论 原代树鼩肝细胞体外可被HCV感染且支持病毒复制。     

人肝癌细胞株7721HCV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HCV体外长期复制的感染细胞模型。方法将慢性丙型肝炎患者的血清与人肝癌细胞系7721共同孵育8小时后,分别以反转录-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正、负RNA、HCV抗原的表达及HCV-RNA在感染细胞内的定位。结果感染血清和细胞共同孵育后的第2～65天,从细胞内和培养上清中均可间断地检测出HCV正、负RNA,即使其间细胞传代4次HCV NS_3抗原、NS_5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、复制机理的研究、抗病毒药物的评价及保护性抗体/疫苗的初步筛选。     

痘苗病毒辅助的重组丙型肝炎病毒生产体系中生产细胞的选择

目的:探讨痘苗病毒辅助的重组丙型肝炎病毒制备体系中生产细胞的选择.方法:将已构建好的质粒pT7HCV及pVHCV分别共转染BHK21,HepG2,SMMC7721细胞株,4 h后用痘苗病毒感染细胞,96 h后用RT-PCR检测HCV的RNA表达.用荧光定量PCR方法检测培养上清中RNA的拷贝数,并用Western blot检测结构蛋白E2以及非结构蛋白NS5的表达.结果:PT-PCR检测到各细胞株培养上清中均有HCV的RNA的存在,定量PCR显示RNA的拷贝数在BHK21,HepG2,SMMC7721细胞中的产量分别为(2.974±0.8)×107,(1.99±0.17)×105,(1.19±0.17)×105(copies/mL).Western blot检测到在这3种生产细胞中均有HCV非结构蛋白NS5的表达.结论:BHK21,HepG2,SMMC7721细胞株均可生产出重组丙型肝炎病毒颗粒,且以BHK21为生产细胞株时产量最高.     

末梢血单个核细胞被HCV感染

我们调查了丙型肝炎患者末稍血单个核细胞(PBMC)被HCV感染的情况。用逆转录——套式PCR法检测了HCV RNA正、负链。22例受试者的PBMC中检出HCV RNA正链12例(54.5％),其中5例(22.7％)负链也阳性。结果提示,在丙肝患者的PBMC中存在着HCV的活动性感染和复制,表明了HCV的肝细胞外感染。     

慢性丙型肝炎患者外周血单个核细胞中负链HCV RNA的检测及意义

19例临床诊断慢性丙型肝炎患者,检测其外周血单个核细胞中丙型肝炎病毒(HCV)RNA的存在及复制状态。所选检测对象血清标本抗-HCV及HCV RNA均为阳性。采用逆转录-套式PCR法检测其外周血单个核细胞中HCV正、负链RNA,结果19例患者外周血单个核细胞中14例HCV正链RNA阳性;6例HCV负链RNA阳性。实验结果证实部分慢性丙型肝炎患者外周血单个核细胞中存在HCV复制,表明肝细胞并非为HCV感染与复制的唯一场所。HCV感染慢性化与其外周血单个核细胞中存在HCV复制可能有一定关系。     

丙型肝炎病毒肝外组织感染与肝外肿瘤的关系

目的：研究丙型肝炎病毒（HCV）在肝外组织感染及复制状况，探讨HCV与肝外肿瘤的关系。方法：采用免疫组化技术对70例慢性丙型肝炎（CHC）患者合并外科疾患的手术切除标本进行HCV抗原标记；RT-PCR检测组织中HCV正、负链RNA。结果：70例CHC合并恶性肿瘤的组织标本37份，HCV抗原阳性率为67.6％（25／37）；非恶性肿瘤的组织标本33份，HCV抗原阳性率为24．2％（8／33），两者比较差异有显著性意义（Х^2=13．14，P〈0．05）。33份HCV抗原阳性的组织中21份（63.6％）检出HCV正链RNA，10份（30.3％）检出HCV负链RNA，且组织中正链和负链HCV RNA的检出与细胞内HCV抗原表达信号呈较好的一致性。结论：HCV可以感染肝外组织，其中部分肝外组织是HCV的复制场所；在CHC患者HCV除诱发原发性肝癌外，还可能诱发其他的恶性肿瘤。     

丙型肝炎病毒感染人单核细胞系U937的研究

利用HCV RNA阳性血清感染人类单核细胞（U937），并利用RT－PCR测定感染后的产物，于感染后的第2天、第4天和第7天发现HCV RNA的正、负链。结果表明，采用本方法可以建立HCV RNA体外感染人类单核细胞的模型，并有病毒复制现象。     

Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

AIM To establish a cell culture system with long-termreplication of hepatitis C virus in vitro.METHODS Human hepatoma cell line 7721 was tested forits susceptibility to HCV by incubating with a serum from apatient with chronic hepatitis C.Cells and supernatantwere harvested at various phases during the culturingperiods.The presence of HCV RNA,the expression ofHCV antigens in cells and/or supernatant were examinedby RT-PCR,in situ hybridization and immunohisto-chemistry respectively.RESULTS The intracellular HCV RNA was first detectedon d2 after infection and then could be intermittentlydetected in both cells and supernatant over a period of atleast three months.The expression of HCV NS_3,CP_(10)antigens could be observed in cells.The fresh cells couldbe infected by supernatant from cultured infected cellsand the transmission of viral genome from HCV-infected7721 cells to PBMCs was also observed.CONCLUSION The hepatoma line 7721 is not onlysusceptible to HCV but also supports its long-termreplication in vitro.     

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。     

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.     

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

AIM: To establish a cell culture system with long-term 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 El antigens in cells were examined by RT-PCR and immunological techniques (flow cytom-etry 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-El). 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.     

Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells

AIM： To study the expression and phosphorylation of extracellular signal-regulated kinase （ERK） i and ERK2 in multidrug resistant （MDR） hepatocellular carcinoma （HCC） cells.
METHODS： MDR HCC cell lines, HepG2/adriamycin （ADM） and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein （P-gp） and multidrug resistant protein 1 （MRP1） expression levels. ERK1 and ERK2 mRNA expression lev-ls were measured by quantitative real-time PCR （QRTPCR）. Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.
RESULTS： MTT assay showed that HepG2/ADM andSMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells （8.92% ±0.22% vs 0.88% ± 0.05%, P 〈 0.001） and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells （7.37% ± 0.26% vs 1.74% ± 0.25%, P 〈 0.001）. However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.
CONCLUSION： ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells,     

miR-155在肝细胞癌中的表达及对肝癌细胞增殖的影响

目的:检测微小RNA-155(miR-155)在肝癌组织中的表达并分析其对肝癌细胞增殖和细胞凋亡的影响.方法:采用TagMan MGB探针法荧光定量P C R分析42例原发性肝癌及对应的癌旁组织miR-155的表达;利用miR-155反义寡核苷酸(ASO-miR-155)降低肝癌细胞HepG2和SMMC7721中miR-155的表达;利用MTT比色法检测肝癌细胞增殖的变化,并通过流式细胞技术检测肝癌细胞早期凋亡情况.结果:42例肝癌及癌旁组织标本中,miR-155在52%(22/42)肝癌组织中的表达明显高于癌旁组织(P<0.05);利用脂质体将ASO-miR-155转染肝癌细胞HepG2和SMMC7721后,miR-155的表达明显降低,肝癌细胞HepG2和SMMC7721生长受到明显抑制;并且细胞的早期凋亡明显增加.结论:miR-155在肝癌组织中过表达,降低其表达能明显抑制肝癌细胞的生长并诱导细胞早期凋亡,miR-155有可能成为肝癌治疗的新靶点.     

母体表达基因3通过miR-125a-5p/TET2途径抑制HepG2细胞载脂蛋白(a)的表达

目的研究发现母体表达基因3(MEG3)对HepG2细胞载脂蛋白(a)[Apo(a)]表达的调控作用及其机制。方法用荧光素酶报告系统分析MEG3与miR-125a-5p的靶向性结合。采用实时定量PCR(qRT-PCR)检测高表达Apo(a)的HepG2细胞和低表达Apo(a)的SMMC7721细胞中MEG3的表达情况;向HepG2细胞转染MEG3,Western blot和qRT-PCR检测Apo(a)、TET2表达情况;采用小干扰RNA技术沉默TET2的表达。结果 (1)MEG3与hsa-miR-125a-5p能互补性结合,荧光素酶报告基因系统分析结果证实了MEG3与hsa-miR-125a-5p结合的存在。(2)miR芯片结果表明,在HepG2细胞中,hsa-miR-125a-5p表达水平升高,是对照组的近1.5倍,MEG3在HepG2细胞和SMMC7721细胞中均有表达,但前者MEG3的表达水平显著低于后者。(3)MEG3抑制Apo(a)表达。(4)MEG3下调miR-125a-5p的表达,上调TET2的表达; miR-125a-5p的mimics可逆转MEG3对Apo(a)的下调作用及TET2的表达,但可被miR-125a-5p的抑制剂逆转; TET2沉默可逆转MEG3对Apo(a)的下调作用。结论 MEG3通过miR-125a-5p/TET2途径下调HepG2细胞Apo(a)的表达。     

Growth inhibition and apoptosis induction of Sulindac on Human gastric cancer cells

AIM: To evaluate the effects of sulindac in inducing growth inhibition and apoptosis of human gastric cancer cells in comparison with human hepatocellular carcinoma (HCC) cells. METHODS: The human gastric cancer cell lines MKN45 and MKN28 and human hepatocellular carcinoma cell lines HepG(2) and SMMC7721 were used for the study. Anti-proliferative effect was measured by MTT assay, and apoptosis was determined by Hoechst-33258 staining, electronography and DNA fragmentation. The protein of cyclooxygenase-2 (COX-2) and Bcl-2 were detected by Western dot blotting. RESULTS: Sulindac could initiate growth inhibition and apoptosis of MKN45, MKN28, HepG(2) and SMMC7721 cells in a dose-and time-dependent manner. Growth inhibitory activity and apoptosis were more sensitive in HepG(2) cells than in SMMC7721 cells, MKN45 and MKN28 cells. After 24 hours incubation with sulindac at 2mmol x L(-1) and 4mmol x L(-1), the level of COX-2 and Bcl-2 protein were lowered in MKN45, SMMC7721 and HepG(2) cells but not in MKN28 cells. CONCLUSION: Sulindac could inhibit the growth of gastric cancer cells and HCC cells effectively in vitro by apoptosis induction, which was associated with regression of COX-2 and Bcl-2 expression. The growth inhibition and apoptosis of HCC cells were greater than that of human gastric cancer cells. The different effects of apoptosis in gastric cancer cells may be related to the differentiation of the cells.