柯萨奇病毒A组6型敏感细胞系的筛选与验证  

Screening and validation of sensitive cell lines for coxsackievirus A6

作　　者：周雨晴 王筱 张胤雯 赵冰[1] 张雪纯 郝莉鹏 潘丽峰[1,2] ZHOU Yuqing;WANG Xiao;ZHANG Yinwen;ZHAO Bing;ZHANG Xuechun;HAO Lipeng;PAN Lifeng(Microbiology Laboratory,udong Center for Disease Control and Prevention,Shanghai 200136,China;不详)

机构地区：[1]上海市浦东新区疾病预防控制中心微生物实验室,上海200136 [2]复旦大学浦东预防医学研究院,上海200136

出　　处：《中国病毒病杂志》2024年第6期555-562,共8页Chinese Journal of Viral Diseases

基　　金：上海市卫生健康委员会卫生行业临床研究专项(202240094);上海市浦东新区卫生健康委员会公共卫生高原学科建设计划项目(PWYggy2021-01);上海市浦东新区疾病预防控制中心科研项目(PDCDC-2022-02)。

摘　　要：目的筛选柯萨奇病毒A组6型(coxsackievirus A6,CVA6)的敏感细胞系,为毒株分离及后续病毒机制研究与疫苗候选株筛选提供合适的细胞基质。方法将CVA6毒株分别感染非洲绿猴肾(African green monkey kidney,Vero)细胞、人胚肺二倍体(human embryo lung diploid cells,KMB17)细胞、恶性胚胎横纹肌瘤(human rhabdomyosarcoma,RD)细胞和人喉癌上皮细胞(human larynx epidermoid carcinoma cells,Hep-2细胞),光学显微镜观察细胞病变效应,以病变特点、半数组织培养感染剂量(median tissue culture infective dose,TCID50)进行敏感细胞筛选。以细胞形态、活力、生长曲线、传代稳定性、病毒敏感性为检测指标验证Vero细胞为基质的CVA6毒株的适应性。结果4种细胞对CVA6的敏感性依次为Vero>KMB17>RD>Hep-2,其中以Vero细胞开始病变时间最快,感染性滴度最高(3.777 lgTCID50/ml),可作为CVA6疫苗候选株的理想基质。所用工作细胞种子为复苏后24 h活率为98.97%,呈梭形上皮样形态,铺路石样排列,生长曲线呈S形,在72~168 h进入生长对数期,最大细胞浓度为6.93×106个/ml,群体倍增时间为25 h,细胞连续传10代增殖速度稳定且细胞形态正常,CVA6与CVA16毒株对Vero细胞敏感性一致。结论Vero细胞是CVA6感染的4种不同细胞中最敏感的细胞,可作为CVA6毒株分离及后续疫苗筛选的首选细胞。Objective To screen and identify the sensitive cell lines for coxsackievirus A6(CVA6),providing a suitable cellular matrix for strain isolation,subsequent viral mechanism studies,and vaccine candidate screening.Methods The CVA6 strains were used to infect African green monkey kidney(Vero)cells,human embryo lung diploid cells(KMB17)cells,human rhabdomyosarcoma(RD)cells,and human larynx epidermoid carcinoma(Hep-2)cells,respectively.The cytopathic effects(CPE)were observed under an optical microscope to identify sensitive cell lines based on the characteristics of cell damage and median tissue culture infective dose(TCID50).The sensitivity of CVA6 to Vero cells was further validated for their suitability as a substrate for CVA6 based on cell morphology,viability,growth curves,passage stability,and viral sensitivity.Results The sensitivity of different cells to CVA6 was ranked as follows:Vero>KMB17>RD>Hep-2.Vero cells exhibited the earliest onset of cytopathic effects and the highest infectious titer(3.777 lg TCID50/ml),making them the most suitable substrate for CVA6 vaccine candidates.The working cell line had a viability of 98.97%24 hours post-recovery,displayed spindle-shaped epithelial morphology with a cobblestone arrangement,and exhibited an S-shaped growth curve.The logarithmic growth phase was reached between 72 and 168 hours,with a maximum cell concentration of 6.93×106 cells/ml and a population doubling time of 25 hours.The proliferation rate remained stable over ten passages,with normal cell morphology,and both CVA6 and CVA16 strains showed consistent sensitivity in Vero cells.Conclusion Vero cells are the most sensitive among the four cell lines for CVA6 infection and are recommended as the primary cell line for strain isolation and vaccine candidate screening.

关 键 词：VERO细胞 柯萨奇病毒A组6型 RD细胞 KMB17细胞 HEP-2细胞 

分 类 号：R512.5[医药卫生—内科学]