通过原位逆转录聚合酶链反应检测体外经爱泼斯坦-巴尔病毒转化的人B淋巴细胞中的丙型肝炎病毒
Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction.
作者信息
Cheng J L, Liu B L, Zhang Y, Tong W B, Yan Z, Feng B F
机构信息
Institute of Hepatology, People's Hospital, Medical Center of Beijing, University of Beijing, China.
出版信息
World J Gastroenterol. 2001 Jun;7(3):370-5. doi: 10.3748/wjg.v7.i3.370.
AIM
To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro.
METHODS
Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR.
RESULTS
HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion.
CONCLUSION
HCV may exist and remain functional in a cultured cell line for a long period.
目的
研究丙型肝炎病毒（HCV）在体外培养的患者外周血单个核细胞（PBMC）中的持续性和复制情况。
方法
利用爱泼斯坦-巴尔病毒（EBV）将一名HCV阳性患者的丙型肝炎病毒转化为永久性淋巴母细胞系（LCL）。每月通过逆转录聚合酶链反应（RT-PCR）检测培养细胞和生长培养基中的HCV正链和负链RNA。采用免疫组织化学SP法和原位RT-PCR进一步检测HCV的核心蛋白和NS5蛋白。
结果
在培养的细胞中持续检测到HCV RNA正链达一年时间。LCL细胞中的负链RNA和上清液中的正链RNA呈间歇性观察到。免疫组织化学结果显示HCV NS3和C蛋白的表达主要在LCL细胞质中。PCR产物的阳性信号为深蓝色，主要定位于LCL细胞质中。RT-PCR信号经过夜核糖核酸酶消化消除，但经脱氧核糖核酸酶消化未消除。
结论
HCV可能在培养的细胞系中长期存在并保持功能。
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