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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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