戊型肝炎病毒在人胚肺二倍体细胞KMB17等传代细胞中的繁殖

Propagation of Hepatitis E Virus in Several Cell Lines Including Human Embryo Lung Diploid Cell KMB17

目的探索戊型肝炎病毒(HEV)敏感细胞及体外培养条件,为HEV体外研究提供基础。方法用戊肝患者急性期阳性粪便悬液感染恒河猴进行毒种的扩增与活化,超速离心处理猴阳性粪便标本获取培养用毒种,将其接种于各种人源性细胞(包括人胚肺二倍体细胞KMB17、人肺癌细胞A549、人肝癌细胞BEL7402、人宫颈癌细胞Hela)和非人灵长类细胞(非洲绿猴肾细胞Vero),通过细胞病变观察、RT-PCR检测及免疫荧光实验来判定细胞是否对HEV敏感。结果KMB17、A549、BEL7402细胞中第一代传代7～9d出现细胞病变,11～13d脱落,传至10代仍可经正链和负链RT-PCR检测到HEV基因组正链RNA和复制的负链RNA的存在,而Hela、Vero细胞未见细胞病变,分别于2～4代后经RT-PCR不能扩增到特异片段。在KMB17细胞培养体系中经免疫荧光实验表明实验组有明显荧光着色,病毒捕获RT-PCR扩增到特异片段。结论在采用的培养条件下,KMB17、A549、BEL7402细胞对HEV敏感,而Hela、Vero细胞不敏感,本研究建立了一定代次内HEV组织培养体系。

Objective To investigate the hepatitis E virus(HEV)sensitive cells and its tissue culture condi -tions.Methods The HEV from dejecta supernatant of patients with acute hepatitis E was amplified an d activated by passaged in Rhesus.Then,the positive dejecta samples of infected monkeys were dealed with super-centrifugation and virus for culture was obtained.Various human-derived(including KM B17,A549,BEL7402,and Hela)and non-human primates derived cells(Vero)were inoculated with HEV.Sensitivity of cells to HEV was measured by CPE(cytopathic effect),RT-PCR and immu nofl uorescence.Results CPE in KMB17,A549and BEL7402cells appeared during7～9days,meanwhile,cells shelled during11～13days on the first filial generation.The existence of HEV genome +RNA and replicated-RNA was still detectable by RT-PCR after the tenth filial generation.Neither CPE nor amplification of HEV genome RNA could be detected in Hela and Vero cells after the second to fourth filial generation.HEV could also be detected from inoculated KMB17cells by im mu nofluorescence and RT-PCR.Conclusions It indicates that KMB17,A549and BEL7402cells are sensitive to HEV under the experimental culture conditions,while Hela and Vero cells are insensitive.Tissue culture system of HEV in certain filial generation is established.