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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 investigate the way of HBV transfection into HepG2 cells and to identify the limited factors that make it difficult for HBV to naturally infect cell lines, so as to lay the foundation for the establishment of a natural HBV infected cell model. Methods HepG2 cells were adsorbed by HBV at 4 ℃ and 37 ℃, and the pancreatic enzyme digestion and intracellular viral DNA were detected by PCR. The other part of HepG2 cells after cryogenic synchronization treatment, after inoculation of HBV, at different time phase were collected cells and culture supernatant, some of the cells were nuclear plasma separation, indirect immunofluorescence, ELISA and PCR were used to detect the virus antigen And DNA, and HepG2 cell HBVcc cDNA was detected by selective PCR. Results Under the condition of 4 ℃, HBV only adsorbed on the surface of HepG2 cell membrane. At 37 ℃, the virus particles partially adsorbed on the surface of HepG2 cells penetrated into the cytoplasm, and HBV DNA could be detected in HepG2 cells and trypsin solution. HepG2 cells were positive for HBV DNA at 4 h, 8 h and 24 h after vaccination, weakly positive at 48 h, and negative at 96 h. Supernatant HBV DNA and virus antigens in each phase were negative. After HBV penetrated into HepG2 cells, it was mainly distributed in the cytoplasm first, followed by nucleus aggregation. The virus in the cytoplasm gradually decreased until it disappeared (24 h after infection), and the number of nuclei in the nucleus gradually increased from 48 h began to decline, disappear to 96 h. Virus DNA was detected in nuclear membrane and nuclear plasma 8 h after virus inoculation. Each phase of cccDNA test has always been negative. Conclusion HBV may infect target cells in “co-receptor” mode. Blocking of virus shedding may be the main limiting factor for HepG2 cells not supporting HBV replication.