HBV感染HepG2细胞早期过程研究

Early events of hepatitis B virus infection of HepG2 cells

目的 探讨HBV吸附穿入HepG2细胞的方式,明确HBV难以自然感染细胞系的受限因素,为建立HBV自然感染细胞模型奠定基础。方法 在4℃和37℃条件下,HBV吸附HepG2细胞,用PCR方法检测胰酶消化液和细胞内的病毒DNA。另一部分HepG2细胞经过低温同步化处理,接种HBV后,于不同时相分别收集细胞和培养上清液,其中部分细胞进行核浆分离,通过间接免疫荧光、ELISA和PCR方法分别检测其中的病毒抗原和DNA,并且通过选择性PCR检测HepG2 细胞HBVcccDNA。结果 在4℃条件下,HBV只是吸附于HepG2细胞膜表面。37℃条件下,部分吸附于HepG2细胞表面的病毒颗粒发生穿入,进入细胞浆,HepG2细胞和胰酶溶液中均可检出HBV DNA。接种病毒后4 h、8 h、24 h HepG2细胞HBV DNA均呈阳性,至48 h转为弱阳性,而96 h为阴性。各时相上清液HBV DNA和病毒抗原均为阴性。HBV穿入HepG2细胞后,首先主要分布于胞浆中,随后出现胞核聚集性,胞浆中的病毒逐渐减少直至消失(感染后24 h),而胞核中的病毒数量早期逐渐增加,从48 h开始呈下降趋势,至96 h消失。病毒接种后8 h核膜与核浆中均可检出病毒DNA。各时相cccDNA检测始终阴性。结论 HBV可能以“共受体”模式感染靶细胞。病毒脱衣壳过程受阻可能是HepG2细胞不支持HBV复制型感染的主要受限因素。

Objective To explore the mode of hepatitis B virus (HBV) adsorption and penetration into HepG2 cells, and to identify the limiting factors of HBV natural infection infinite cell line. Methods HBV adsorbed to HepG2 cells at 4℃ or 37℃, then HBV DNA within trypsinization solution and HepG2 cells were detected by polymerase chain reaction (PCR). After synchronization of cells at low temperature, another group of HepG2 cells were inoculated with HBV. Subsequently, culture supernate and cells were collected at various phases. Aliquots of collected cells were partitioned into nuclear and cytoplasmic components. HBV DNA and antigens were detected by PCR and enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence (IIF). HBV covalently closed circular DNA (cccDNA) was examined by selective PCR. Results HBV was merely able to bind to HepG2 cells surface at 4℃. However, HBV could penetrate into cytoplasm at 37℃ and HBV DNA was positive both in trypsinization solution and HepG2 cells by PCR. After incubation at 37℃, HBV DNA could be detected in HepG2 cells at 4?h-24?h, however, viral DNA became weakly positive and negative at 48?h and 96?h, respectively. Antigens and DNA of HBV in culture supernate had been negative after incubation at 37℃ and HBV congregated in nucleus. Viral DNA within the nucleus increased from 4?h to 24?h after incubation, but became weakly positive at 48?h, and disappeared at 96?h. HBV DNA was detected in both nucleoplasm and nuclear membrane at 8?h after inoculation. cccDNA was all negative. Conclusion HBV infected HepG2 cells by co-receptors mode. The failure of uncoating viral genome maybe the main limiting factor that lead to HepG2 cells retractile to HBV replicative infection.