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Detection of hepatitis C virus in the supernatant of human placenta trophoblastic cells and cell lysates
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摘要: 目的应用改良的分离、纯化方法对孕早期人绒毛滋养细胞进行原代培养,利用HCV阳性血清对滋养细胞进行体外感染,探讨HCV在体外培养条件下对细胞的感染及HCV在细胞中的复制状态。方法采用胰蛋白酶消化法消化正常人妊娠孕早期胎盘组织,以35%、45%2个Percoll密度梯度进行分离纯化、并免疫组化鉴定,获得纯度较高的滋养层细胞,应用HCV阳性且高载量的血清进行体外感染,应用免疫荧光染色检测滋养层细胞中HCV NS5表达,通过细胞裂解液裂解感染后细胞,应用荧光定量PCR（RT-PCR）技术检测感染后细胞培养上清和细胞内的HCV RNA,以观察HCV的感染及复制情况。结果该法分离纯化的滋养层细胞生长良好,特异性角蛋白-7阳性,纯度高。原代滋养层细胞在与HCV阳性血清共同孵育后均未出现明显的细胞病变,感染后48 h在滋养层细胞细胞浆中可见HCV NS5表达,感染后分别于24、48、72、96、120 h收集的培养上清和感染后细胞内均可以检测到HCV RNA。排除了培养洗涤液HCV污染,进一步检测其细胞裂解液中HCV,结果检测HCV阳性。结论利用改良后人胎盘滋养层细胞的体外培养系统,HCV阳性血清能在...Abstract: Objective To investigate HCV infection and replication in the primarily cultured human trophoblastic cells which were cultured by improved separation and purification methods and co-cultured with HCV infection serum.Methods Human placenta was digested with trypsin-DNase and then centrifuged with percoll density gradient in order to obtain the trophoblastic cells, which were incubated in HCV positive serum (the quantification of HCV RNA was 5.9×107copies/ml) .The expression of HCV non-structure protein 5 (HCV-NS5) was evaluated by immunofluorescence.The infected cells were lysed.HCV RNA in HCV infected trophoblast cells was quantitated by RT-PCR.Results The culture model of trophoblastic cells from human placenta was established and cytokerat was observed in positive cells.Observation of the morphology of trophoblastic cells showed that no significant cytopathic effect was found when co-cultured with HCV positive serum.The antigen of HCV NS5 could be detected at 48 hours after infection.The HCV RNA was detected in the supernatent of the culture medium at 24, 48, 72, 96, 120 hours after infection respectively.HCV RNA was detected in HCV infected cells.Washing liquid was not contaminated by HCV positive serum.Cell lysates which contained HCV RNA was detected by RT-PCR.Conclusion Our study indicates that trophoblastic cells cultured in vitro can be infected by HCV.The replication of HCV in trophoblastic cells was detected.
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Key words:
- hepacivirus /
- trophoblastic
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[1]Soriano V, Vispo E, Poveda E, et al.Directly acting antivirals against hepatitis C virus[J].J Antimicrob Chemother, 2011, 66 (8) :1673-1686. [2]Sievert W, Altraif I, Razavi HA, et al.A systematic review of hepatitis C virus epidemiology in Asia Australia and Egypt[J].Liver Int, 2011, 31 (2) :61-80. [3]Ghamar Chehreh ME, Tabatabaei SV, Khazanehdari S, et al.Effect of cesarean section on the risk of perinatal transmis-sion of hepatitis C virus from HCV-RNA+/HIV-mothers:a meta-analysis[J].2011, 283 (2) :255-260. [4]聂青和.丙型肝炎病毒母婴感染研究及其预防现状 (述评) [J].世界华人消化杂志, 2005, 13 (11) :1257-1262. [5]程勇前, 聂青和, 周永兴.丙型肝炎病毒母婴传播机制研究[J].世界华人消化杂志, 2002, 10 (4) :445-447. [6]张亚飞, 聂青和, 罗新栋, 等人滋养层细胞内丙型肝炎病毒样颗粒的免疫电镜形态学研究[J].医学研究生学报, 2005, 18 (8) :674-681. [7]程勇前, 聂青和, 周永兴, 等.透射电镜及激光共聚焦技术观察体外丙型肝炎病毒感染的人胎盘滋养层细胞[J].世界华人消化杂志, 2003, 11 (2) :151-156. [8]程勇前, 聂青和, 周永兴, 等.体外感染丙型肝炎病毒的滋养层细胞超微结构变化[J].中华妇产科杂志, 2003, 38 (1) :28-29. [9]聂青和, 张亚飞, 罗新栋, 等.体外培养人胎盘滋养层细胞内吞丙型肝炎病毒过程的电镜观察[J].中华传染病杂志, 2005, 23 (6) :364-367. [10]程勇前, 聂青和, 周永兴, 等.人滋养层细胞分离培养及HCV体外感染试验[J].第四军医大学学报, 2002, 23:1544-1547. [11]聂青和, 程勇前, 黄晓峰, 等.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.实用肝脏病杂志[J].2010, 13 (2) :81-84. [12]Mast EE, Hwang LY, Seto DS, et al.Risk factors for perina-tal transmission of Hepatitis C virus (HCV) and the natural history of HCV infection acquired in infancy[J].Infect Dis, 2005, 192 (11) :1880-1889. [13]聂青和, 王平忠, 周永兴.丙型肝炎病毒感染孕妇羊水中HCV RNA检测的临床意义[J].中华妇产科杂志, 2002, 37 (1) :19-21. [14]Syriopoulou V, Nikolopoulou G, Daikos GL, et al.Mother to child transmission of hepatitis C virus:rate of infection and risk factors[J].Scand J Infect Dis, 2005, 37 (5) :350-353. [15]张亚飞, 聂青和, 邵彬, 等.丙型肝炎病毒持续感染对体外培养人胎盘合体滋养层细胞生物学性状的影响[J].2006, 19 (2) :114-116. [16]Wang YZ, Wang WB, Cao MM, et al.Function of nonstruc-tural5A protein of genotype2a in replication and infection of HCV with gene substitution[J].World J Gastroenterol, 2011, 17 (29) :3398-3406. [17]Indolfi G, Resti M.Perinatal transmission of hepatitis C virus infection[J].J Med Virol, 2009, 81 (5) :836-843. -
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