CAJ | 학술논문

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、CD20抗原阳性，CD21分子消失。（2）传代培养细胞中HCV RNA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。HCV RNA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、HCV RNA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。
목적관찰병형간염병인외주혈B세포장기존활상태하기중시부잉유병형간염병독（HCV）존재，탐토건립HCV체외복제세포모형적가능성。방법이용EB병독전화B림파세포기술，건립병형간염환자외주혈단개핵세포（PBMC ）HCV양성전대세포주（LCL），병응용세포배양、염색체현시、류식세포형광염색、역전록-취합매련반응（RT-PCR）、면역조화、원위RT-PCR급전경등기술연구기분자생물학화면역학특성。결과（1） LCL세포핵형47，XX，+mar，세포표면CD19、CD20항원양성，CD21분자소실。（2）전대배양세포중HCV RNA정련지속12개월양성，이배양상청중칙정간단성양성，무명현규률。HCV RNA부련재LCL세포중야정간단양성。HCV기인분형위Ⅱ형주。（3）면역조화、원위PCR화전경발현HCV항원단백、HCV RNA화병독과립균존재우LCL세포장중；전경발현HCV병독과립정원구형，쌍층막결구，정위우세포포질공포내。직경다위45～70nm，개별위110nm。결론 HCV가이재EB병독전화병인B세포주중장기존활、복제화분비。
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.