MDCK、HEp-2、Vero细胞混合冻存适用于分离培养常见病毒研究  

Mixed cryopreservation of MDCK,HEp-2 and Vero for research of common virus isolation culture

作　　者：汪千力[1] 居丽雯[1] 蒋露芳[1] 蔡韵[1] 高颖阳[2] 姜庆五[1] 

机构地区：[1]复旦大学公共卫生学院流行病学教研室,公共卫生安全教育部重点实验室,上海200032 [2]上海市闵行区疾病预防控制中心,上海201101

出　　处：《中华疾病控制杂志》2012年第10期827-832,共6页Chinese Journal of Disease Control & Prevention

基　　金："十一五"国家科技重大专项(2009ZX10004-104);上海市科委资助项目(09DZ1906605)

摘　　要：目的建立犬肾细胞(MDCK)、人喉表皮癌细胞(HEp-2)和非洲绿猴肾细胞(Vero)3种细胞(MHV)混合细胞瓶冻存复苏方法,经冻存复苏的混合细胞用于分离常见呼吸道和肠道病毒,建立病毒性病原检测的方法学平台。方法对数生长期的MDCK、HEp-2和Vero细胞等量保存在含8%二甲基亚砜(dimethyl sulfoxide,DM-SO)冷冻保存液中,液氮经冻存4个月后复苏并进行细胞计数;在复苏后0 h、24 h和48 h倒置显微镜下观察细胞形态、细胞汇合;并用于分离培养常见呼吸道、肠道病毒,观察致细胞病变效应,采用直接与间接免疫荧光法鉴定。结果在液氮中保存4个月的MHV混合细胞,细胞复苏存活率超过99.20%,镜下细胞折光度好,细胞形态良好,均见到相应的致细胞病变效应和免疫荧光检测阳性结果。结论 MDCK、HEp-2和Vero细胞等量保存在含8%DM-SO冷冻保存液中液氮保存4个月复苏存活率高。混合细胞直接冻存复苏方法建立,为常见的病毒性病原分离培养提供了宽泛的合适细胞宿主,保证了病毒性传染病的快速诊断、实验室病毒性传染病监测的可操作性。Objective To establish the mixed cryopreservation method of MHV mixed cells,which include MDCK（ATCC,CCL-34）,HEp-2（ATCC,CCL-23）and Vero（ATCC,CCL-81）.MHV cells after cryopreservation were used to isolate common respiratory viruses and enteroviruses.Establish a methodology platform of viral pathogens detection.Methods Equivalent MHV cells in exponential phase preserved in 8% DMSO cryoprotectant were stored into liquid nitrogen tank.After 4 months,Anabiosis MHV were detected by trypan-blue staining.While in anabiosis and after 0h,24h,48h,cell morphology and confluence were detected under the inverted microscope.And for the isolation and culture of common respiratory viruses,Adenovirus,Enteroviruses,the MHV were observed cytopathic effect by direct/indirect immunofluorescence.Results After 4 months cryopreservation,the rate of cells survival was more than 99.20%.The refractive under microscope and morphology of cell were good.Inoculated with virus-contaminated simulated throat swab specimens,corresponding cytopathic effect and positive results of immunofluorescence were observed.Conclusions The rate of cell survival is ideal after 4 months cryopreservation in an appropriate concentration of DMSO.The establishment of MHV cells cryopreservation and anabiosis provides a broad and suitable host cells for common viral pathogens＇ isolation.It made rapid diagnosis and laboratory surveillance of viral diseases feasible.

关 键 词：上皮样细胞 肠道病毒感染 传染病 

分 类 号：R512.5[医药卫生—内科学]