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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 investigate whether hepatitis C virus (HCV) still exists in the long-term survival of peripheral blood B cells in patients with hepatitis C and to explore the possibility of establishing an in vitro replicating cell model of HCV. Methods HCV positive cell line (LCL) of peripheral blood mononuclear cells (PBMCs) was established by Epstein-Barr virus transformation of B lymphocytes. Cell culture, chromosomal display, flow cytometry, reverse transcription polymerase Chain reaction (RT PCR), immunohistochemistry, in situ RT PCR and electron microscopy and other techniques to study its molecular biology and immunological characteristics. Results (1) LCL karyotype 47, XX, + mar, cell surface CD19, CD20 antigen-positive, CD2 1 molecules disappear. (2) The positive strand of HCVRNA in subculture cells persisted for 12 months, while the supernatant of HCVRNA showed intermittent positive, no obvious regularity. The negative strand of HCV RNA is also intermittent positive in LCL cells. HCV genotypes are type II strains. (3) Immunohistochemistry, in situ PCR and electron microscopy showed that the HCV antigen protein, HCV RNA and virus particles were present in the cytoplasm of LCL. The electron microscopy showed that the virus particles were globular and bilayer membrane structure located in the cytoplasm of the cytoplasm Bubble inside. Most of the diameter of 45 ~ 70nm, individual 110nm. Conclusion HCV can survive, replicate and secrete long-term in B cell line of Epstein-Barr virus transformation patients.