摘要
目的:建立一新型细胞株,使该细胞株既能自然感染HBV又能传代培养,评价杂交细胞对HBV的自然感染能力.方法:分离培养未携带HBV的人原代肝细胞,与诱导突变的HepG2细胞进行融合得杂交细胞,经HAT培养基筛选,利用胰蛋白酶G显带技术鉴定所得细胞,用含HBV的慢性乙型肝炎患者血清感染杂交细胞(同时感染HepG2作为对照),巢式PCR检测感染后细胞内HBVDNA合成及分泌情况及有无HBV复制中间产物HBVcccDNA,间接免疫荧光检测感染后细胞内HBcAg的表达,电化学发光法检测感染后细胞培养上清中的HBsAg和HBeAg.结果:成功建立人原代肝细胞与HepG2的杂交细胞,能体外传代培养,染色体核型分析示杂交细胞染色体众数为99条,证实为融合细胞,HBV感染后第4天起,杂交细胞内和培养上清中均能检测到HBVDNA,HBV感染后第3天起,杂交细胞内可以检测到HBV的复制中间产物HBVcccDNA,HBV感染后第4天起,杂交细胞胞质及部分胞核内HBcAg始终是阳性表达,间接免疫荧光染色呈胞质弥漫性着色,电化学发光法检测培养上清中HBsAg及HBeAg持续表达;而感染后的HepG2细胞检测结果均为阴性.结论:成功建立的兼具有人原代肝细胞和HepG2遗传特性的杂交细胞株可以被慢性乙型肝炎患者血清中的HBV病毒自然感染,可进一步用作研究HBV感染的体外细胞模型.
AIM:To establish a hybrid cell line (by fusing HepG2 cells with primary human hepatocytes) that can be infected by hepatitis B virus (HBV) and be serially subcultured in vitro,and to evaluate the infection ability of HBV in this hybrid cell line.METHODS:Normal human hepatocytes were isolated and cultured.Primary human hepatocytes were then fused with HGPRT-deficient HepG2 cells (induced with ethyl methanesulfonate).The hybrid cells were identifi ed by the trypsin G-banding method.After the hybrid cells and normal HepG2 cells were infected with serum-derived HBV virions,intracellular and secreted HBV DNA as well as intracellular HBV cccDNA (covalent closed circle DNA) were detected by nested polymerase chain reaction (PCR).HBcAg in infected cells was analyzed by indirect immunofluorescence.HBsAg and HBeAg in the supernatants of infected cells were identif ied by electrochemiluminescence.RESULTS:A hybrid cell line was established successfully by fusing HepG2 cells with primary human hepatocytes.This hybrid cell line could be subcultured in vitro.Karyotype analysis showed that the modal chromosome number of hybrid cells was 99.HBV DNA was detected consistently in both hybrid cells and their culture medium 4 days post-infection.HBV cccDNA was detected consistently 3 days post-infection.HBcAg,HBsAg and HBeAg were also detected consistently 4 days post-infection.In contrast,negative results were obtained in control HepG2 cells infected with HBV virions.CONCLUSION:A new hybrid cell line that can be used for establishing an in vitro cell model of HBV infection is established successfully.This new hybrid cell line inherits the characteristics of both HepG2 cells and primary human hepatocytes.
出处
《世界华人消化杂志》
CAS
北大核心
2010年第8期755-760,共6页
World Chinese Journal of Digestology