el-Awady Mostafa K, Tabll Ashraf A, el-Abd Yasmine S, Bahgat Mahmoud M, Shoeb Hussein A, Youssef Samar S, Bader el-Din Noha G, Redwan el-Rashdy M, el-Demellawy Maha, Omran Moataza H, el-Garf Wael T, Goueli Said A
Department of Biomedical Technology, National Research Center, Tahrir Street, PO 12622, Dokki, Cairo, Egypt.
World J Gastroenterol. 2006 Aug 14;12(30):4836-42. doi: 10.3748/wjg.v12.i30.4836.
To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro.
HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively.
The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells.
HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.
建立一种能在体外长期复制丙型肝炎病毒（HCV）基因组并表达病毒抗原的细胞培养系统。
用慢性丙型肝炎患者的血清孵育HepG2细胞系，检测其对HCV的易感性。在培养过程中的不同时间点收获细胞和上清液。检测培养上清液感染未感染细胞的能力。分别通过逆转录-聚合酶链反应（RT-PCR）和免疫技术（流式细胞术和蛋白质印迹法）检测细胞中负链（反义）RNA链的存在以及核心抗原和E1抗原。
感染后第3天首次检测到细胞内HCV RNA，随后在至少三个月的时间内，细胞和上清液中均可持续检测到。新鲜细胞可被培养的感染细胞的上清液感染。流式细胞术分析显示，使用自制的多克隆抗体（抗核心抗体和抗E1抗体）可检测到表面和细胞内HCV抗原表达。蛋白质印迹分析显示，在感染细胞培养一个月时，分子量在31至45 kDa之间的一簇免疫原性肽表达，而在未感染的HepG2细胞中未检测到该簇肽。
HepG2细胞系不仅对HCV感染敏感，而且在体外支持其复制。在感染的HepG2细胞中可检测到HCV结构蛋白的表达。这些细胞还能够将病毒颗粒释放到培养基中，而这些病毒颗粒又会感染未感染的细胞。
