禽流感病毒感染K-562和HL-60肿瘤细胞系的实验研究  

Exprerimental study of K-562 and HL-60tumor cell lines infected by avian influenzs vinus

作　　者：张红梅[1] 赵丹[1] 吴玫[1] 牟旭鹏[1] 周伯平[2] 

机构地区：[1]吉林大学中日联谊医院唐敖庆特聘教授实验室,吉林长春130033 [2]深圳市第三人民医院肝病研究所,广东深圳518112

出　　处：《中国实验诊断学》2013年第8期1375-1379,共5页Chinese Journal of Laboratory Diagnosis

基　　金：吉林省自然科学基金201115090

摘　　要：目的选择代表不同骨髓细胞分化阶段的血液肿瘤细胞株K-562、HL-60作为研究对象,选取低致病性禽流感病毒H3N2/H4N6作为病原体,探讨禽流感病毒感染骨髓的机制。方法应用流式细胞术检测K562等细胞表面禽流感病毒受体;收集感染病毒的细胞培养上清,红细胞凝集实验检测染毒细胞释放的子代病毒;收集感染病毒的细胞免疫荧光检测病毒NP,判断病毒吸附、进入细胞并在细胞内增殖、复制情况。结果所检测细胞表面均有禽流感病毒SAα2,3-Gal和SAα2,6-Gal连接的唾液酸寡糖受体的表达。在H3N2感染的K-562细胞培养上清中检测到病毒HA,且随培养时间的延长,病毒的血凝滴度增大。在H4N6感染的K-562细胞的培养上清中病毒HA检测阳性,第三天,K-562细胞上清的病毒血凝滴度即达4,第四天达16;HL-60细胞,只在第三、四天达2;随后荧光显微镜下观察可见,染毒细胞培养0h时,胞膜、胞浆内即可见病毒分布,H4N6感染的细胞荧光强度大于H3N2感染的细胞;H3N2和H4N6感染的细胞中,K562细胞的荧光强度大于其他细胞。随着培养时间的延长,K562细胞表面的病毒增多,而HL-60细胞无明显变化。结论 K-562、HL-60细胞表面均表达禽流感病毒SA-α2,3Gal和SA-α2,6Gal连接的唾液酸寡糖受体。同一病毒H3N2或H4N6的吸附、繁殖和复制及释放情况存在较大差异。Objective Selecting blood tumor cell lines K-562,HL-60which represent different stages of the differentiation of bone marrow cells as the research object,and selecting the low pathogenic avian influenza virus H3N2 / H4N6 as pathogens,to investigate the mechanism of bone marrow infected by avian influenza virus.Methods Flow cytometry was applied to detect avian influenza virus receptors on the surface of K562and other cells;the supernatant of virus-infected cells was collected,Erythrocyte agglutination test was applied to assay progeny virus which was released by infected cells.Immunofluorescence of infected cells was collected to detect virus NP,and then determine the virus adsorption,intracellular proliferation and replication after enters the cell.Results The cells were detected all had expression of avian influenza virus SAα2,3-Gal and SAα2,6-Gal connection with sialic acid oligosaccharides receptor.The detection of hemagglutinining activity in the supernatant of infectious cells showed that the virus was detected only in the supernatant of infected K-562cell after H3N2infection.And the detection of HA in the supernatant of K-562showed positive results among the cells infecting H4N6.The haemagglutinin titer of virus in the supernatant of K-562cell increased to 4at the third day and 16at the fourth day.But that of HL-60cell increased to 2at the third day and the fourth day.It was observed with the microscope that virus distributed on the cell membrane and in the cytoplasm when infected cells were cultured for 0h.The fluorescence intensity of the cells infecting H4N6is stronger than of infecting H3N2.The fluorescence intensity of K-562cells is stronger than other cells among the cells infecting H3N2and H4N6.It indicated that the virus immediately adsorbed to the cell surface and entered the cells after cultivating infected cells for 0h.With the prolonged culture hours,the viruses of the K-562cell surface increased,while HL-60did not change significantly.Conclusion The surface of K-562and HL-60cells all express

关 键 词：禽流感病毒 骨髓 低致病性 受体 植物凝集素 

分 类 号：R511.7[医药卫生—内科学]