丙型肝炎病毒Ⅱ型阳性PBMC永生化细胞株的生物学特性  

Study of biologic characteristics on Epstein-Barr virus transformed B lymphocytes carrying hepatitis C virus type Ⅱ

作　　者：程计林[1] 仝文斌[1] 冯百芳[1] 陶其敏[1] 朱伟 刘宝玲[3] 陈良标[4] 陈佩兰[4] 

机构地区：[1]北京大学医学部人民医院肝病研究所,100044 [2]血液科 [3]河南省焦作市人民医院 [4]北京军区总医院电镜室

出　　处：《中华微生物学和免疫学杂志》2001年第3期334-337,共4页Chinese Journal of Microbiology and Immunology

基　　金：卫生部青年科研基金课题! (98 1 2 69)

摘　　要：目的　观察丙型肝炎病人外周血B细胞长期存活状态下其中是否仍有丙型肝炎病毒(HCV)存在 ,探讨建立HCV体外复制细胞模型的可能性。方法　利用EB病毒转化B淋巴细胞技术 ,建立丙型肝炎患者外周血单个核细胞 (PBMC )HCV阳性传代细胞株 (LCL) ,并应用细胞培养、染色体显示、流式细胞荧光染色、逆转录 聚合酶链反应 (RT PCR)、免疫组化、原位RT PCR及电镜等技术研究其分子生物学和免疫学特性。结果　 (1)LCL细胞核型 47,XX ,+mar,细胞表面CD19、CD2 0抗原阳性 ,CD2 1分子消失。 (2 )传代培养细胞中HCVRNA正链持续 12个月阳性 ,而培养上清中则呈间断性阳性 ,无明显规律。HCVRNA负链在LCL细胞中也呈间断阳性。HCV基因分型为Ⅱ型株。 (3)免疫组化、原位PCR和电镜发现HCV抗原蛋白、HCVRNA和病毒颗粒均存在于LCL细胞浆中 ;电镜发现HCV病毒颗粒呈圆球型 ,双层膜结构 ,定位于细胞胞质空泡内。直径多为 45～ 70nm ,个别为 110nm。结论　HCV可以在EB病毒转化病人B细胞株中长期存活、复制和分泌。Objective To study persistence and replication of hepatitis C virus (HCV) in the patients′ peripheral blood mononuclear cells (PBMC) transformed by Epstein-Barr virus (EBV) in vitro and in order to explore the possibility of establishing a cell line for HCV reproduction. Methods EBV infected peripheral B lymphocytes from one patient with HCV positive of PBMC were transformed into permanent lymphoblastoid cell lines (LCL). The general and special biology characteristics of LCL were studied by cell culture, chromosome banding technique, flow fluorescent-activated cell sorter, reverse transcription-polymerase chain reaction (RT-PCR), in situ PCR, immunohistochemistry and electronic microscopy. Results 1. G-banded karyotype of the LCL was 47, XX, +mar, CD19 and CD20, CD antigens of LCL cell surface were positive and CD21 was not expressed. 2. Positive and negative HCV RNA strands from cultured cells and supernatants were detected by RT-PCR every month. HCV RNA plus strands persisted in the cultured cells for more than one year. It is interesting that the minus-strand RNA in LCL and the plus-strand RNA in supernatants were observed intermittently and HCV genome type of the LCL was type Ⅱ. 3. Immunohistochemical study found that HCV NS3 and C proteins were mostly expressed in LCL cytoplasm. In situ PCR showed that HCV RNA positive signal was mainly located in LCL cytoplasm. Electronic microscopy found HCV spherical virus-like particles with diameter of approximately 45 to 70nm, some particles were 110nm in LCL cytoplasmic vesicles. Conclusion HCV may exist in LCL cultured cells for a longer period and reproduce in and secrete into the media. It offers a strong evidence for persistence of HCV RNA in PBMC of HCV-infected patients.

关 键 词：丙型肝炎病毒 丙型肝炎 外周血单个核细胞 HCVⅡ型阳性 B细胞 生物学特性 

分 类 号：R373[医药卫生—病原生物学]