体外细胞培养中甲肝病毒的连续传代及激光扫描共聚焦成像

Continuous Passage and LSCM Imaging of Propagation of HAV in PLC/ PRF/5 Cell Line

观察甲肝病毒(HAV)在体外细胞培养中连续传代的特点及细胞内病毒荧光的激光扫描共聚焦成像。实验中采用了间接免疫荧光法(IF)与激光扫描共聚焦显微镜(LSCM)结合技术,并与ELISA进行比较。LSCM技术发现将NJ-3株HAV接种于PLC/PRF/5细胞1 h后细胞表面有病毒吸附,2-4 h病毒吸附量增多,4 h后逐步减少。在病毒随细胞连续传代中,至第7代病毒增殖量可达到稳定状态并至少可连续传77代,感染病毒的细胞可达95％以上;细胞传代或病毒收获的最佳周期为5 d;经多次冻存、复苏,仍保持良好的细胞形态和产毒性能。结果表明LSCM技术能更早地检出细胞表面吸附的少量病毒和胞浆内微弱的HAAg荧光,而ELISA则需要较多量病毒才能显示阳性。提示前者更适合于少量病毒时的定性,特别是在初次病毒分离时;后者则适合于病毒量较多时的整体定量。

The character of continuous passage of NJ-3/HAV in vitro in company with passing of PLC/PRF/5 cell line and the laser scanning conlocal imaging of HAV propagation were observed. Technically combining IF and LSCM compared with ELISA in this experiment. By LSCM, a few fluorescence granules were observed at the surface of the cells in 1 h after HAV was inoculated into PLC cells, the number of absorbed granules increased in 2 - 4 h and declined after 4 h. During continuous passage, PLC cells infected by HAV grew well without cytopathie effect. HAAg production was stable in the 7th generation and could go down to more than 77 passages. The optimal period of passage and harvest was 5 day. Conformation and capability of produce HAAg of PLC cells could be well maintained even after being frozen and resuscitated for many times. Tittle of HAV adsorbed in cell surface and feeble HAAg fluorescence in cytoplasm could be observed earlier by LSCM, compared with ELISA, which required more HAAg for positive manifestation. The results suggest that former is more applicable to nature analysis for a small quantity of HAV, particularly in isolation of HAV. The latter is applicable to quantitative analysis of mass HAV.