丙型肝炎病毒高变区基因序列的变化  

Variation of hypervariable regions of the genome of hepatitis C virus in EBV transforming PBMC from patients with hepatitis C

作　　者：程计林[1] 刘宝玲[2] 马文彬[1] 仝文斌[1] 高建恩[1] 冯百芳[1] 陶其敏[1] 魏来[1] 

机构地区：[1]北京医科大学人民医院肝病研究所 [2]河南省焦作市人民医院

出　　处：《中华传染病杂志》2001年第5期292-296,共5页Chinese Journal of Infectious Diseases

基　　金：卫生部青年科研基金资助项目 (98 1 2 69)

摘　　要：目的　观察丙型肝炎患者外周血单个核细胞 (PBMC)体外长期存活状态下是否仍有丙型肝炎病毒 (HCV)存在及其不同时期高变区基因序列的变化。方法　用EB病毒感染并使HCV阳性PBMC转化 ,获得HCV的外周血永生化B细胞 (LCL)。而后 ,每个月应用逆转录 套式 聚合酶链反应 (RT net PCR)检测 1次培养细胞和上清液中的HCVRNA ,并用原位RT net PCR观察HCVRNA在细胞中存在的部位 ;应用基因测序技术分析LCL传代培养 1个月和 9个月后HCV高变区(HVR)核苷酸序列 ,并以原患者半年后血清作对照。结果　经连续 1年检测 ,HCV在传代细胞中持续存在 ,培养细胞中的HCVRNA负链和培养上清液中的HCV呈间断阳性 ;原位PCR发现HCVRNA阳性信号弥散性、簇状和团块状分布于胞浆中 ,细胞膜、核膜和细胞核阴性 ;患者血清HCV核苷酸变异集中在第 1491～ 15 83位核苷酸 (即 384～ 414位氨基酸 )和第 176 1～ 1781位核苷酸 (即 473～479位氨基酸 )两个区域 ,而LCL传代培养 1个月和 9个月后细胞中HCVHVR核苷酸同源性为99 .37%。结论　HCV可以在LCL中较长时间存在 。Objective To study the persistence and replication of hepatitis C virus (HCV) RNA in Epstein Barr virus (EBV) transforming peripheral blood mononuclear cell (PBMC) from patients with he patitis C as well as variation of hypervariable regions (HVR) of the HCV genome. Methods PBMC from one patient with hepatitis C was infected by EBV and then transformed into lymphoblasts capable of being propagated indefinitely. Then, HCV RNA of the cultured cells and supernatants was detected by reverse transcriptase polymerase chain reaction (RT PCR) every month and in situ PCR to identify the location of HCV RNA in the cells. The HVR genome sequences of HCV in the first and the ninth month subculture cells were identified by sequence analysis. Results HCV plus strand RNA could be detected in the cultured cells for as long as 1 year. The HCV plus strand RNA could be identified in supernatants and the minus strand RNA were also observed in the cultured cells intermittently. In situ PCR showed that the blue black positive signals of HCV RNA located mainly in cytoplasmas of EBV transforming B cell but negative in nucleous. The positive signal was not found in negative control cells by in situ RT PCR. HCV HVR genome sequence after half year was found to have two regions of a high degree of variability in 1491 to 1583 nucleotide (384 to 414 amino acid) and 1761～1781 nucleotide (473 to 479 amino acid). But, compared the HCV HVR genome sequence in first month subcultured cells with that in ninth month, there was not significant difference. Conclusion HCV may exist in the cultured cell line for a prolonged period wihtout HVR genome sequence changed.

关 键 词：丙型肝炎病毒 B淋巴细胞 高变区 基因序列 PBMC 

分 类 号：R373.21[医药卫生—病原生物学]