RD细胞库的建立及其相关研究  

Establishment of RD cell bank and related application

作　　者：谢学超 赵丽丽 陈磊[1] 曹慧佼 顾美荣[1] 张改梅[1] XIE Xue-chao;ZHAO Li-li;CHEN Lei;CAO Hui-jiao;GU Mei-rong;ZHANG Gai-mei(Beijing Minhai Biotechnology Co.,Ltd.,Center of Research of Novel Combination Vaccine Engineering Technology in Beijing,Beijing 102600,China)

机构地区：[1]北京民海生物科技有限公司北京市新型联合疫苗工程技术研究中心,北京102600

出　　处：《中国病毒病杂志》2022年第4期277-283,共7页Chinese Journal of Viral Diseases

基　　金：北京市科技计划课题(Z201100005420004)。

摘　　要：目的建立人恶性胚胎横纹肌瘤(human rhabdomyosarcoma,RD)细胞工作细胞库,并对RD细胞工作细胞库应用于肠道病毒灭活疫苗滴度的测定及中和抗体检测的可行性进行相关研究。方法建立工作细胞库,按照《中华人民共和国药典》对其进行检定,包括细胞活力、细胞形态、无菌检查、支原体及细胞外源病毒因子检查等,并对RD细胞生长曲线、传代稳定性及肠道病毒敏感性进行研究。结果实验建立的RD细胞工作细胞库呈梭形、大的多核上皮样细胞形态,有良好的生长状态,细胞活率为98.85%,无菌检查、支原体以及细胞外源病毒因子指标均符合规定。RD细胞生长曲线呈S型,细胞在3~6 d进入对数生长期,而后到达平台期,细胞倍增时间为27 h,连续传20代细胞形态正常。不同代次RD细胞检测肠道病毒71型(enterovirus 71,EV-A71)、柯萨奇病毒A组6型(coxsackievirus 6,CVA6)、柯萨奇病毒A组10型(coxsackievirus 10,CVA10)、柯萨奇病毒A组16型(coxsackievirus 16,CVA16)肠道病毒的滴度均值分别为(8.20±0.07)、(7.84±0.12)、(7.93±0.09)、(7.34±0.16)lgCCID/ml,变异系数(coefficient of variation,CV)在0.88%~2.12%。结论建立的工作细胞库符合《中华人民共和国药典》规定,传代稳定性良好,RD细胞对肠道病毒较为敏感,可以用于肠道病毒疫苗相关病毒滴度的测定及疫苗中和抗体检测.Objective To establish a working cell bank of human rhabdomyosarcoma cells(RD)and to assess the feasibility of its application in the titer determination of inactivated enterovirus vaccine and the detection of neutralizing antibodies.Methods Working cell bank of RD was established.According to the Chinese Pharmacopoeia,the viability,morphology,sterility,mycoplasma,exogenous viral factor of cells in the cell bank were tested.Meanwhile,the cell growth,stability during subculture and sensitivity to enterovirus were evaluated.Results Cells in the established working cell bank were spindle or polygonal shaped epithelial-like cells with good growth status and 98.92%viability.The cells passed the tests of sterility,mycoplasma and exogenous viral factor,and with S-shaped growth curve.After culturing for 3—6 days,the cells entered the logarithmic growth phase,and then to the plateau phase.The doubling time was 27 h.The morphology of cells stayed unchanged after subculture for 20 passages.The mean titers in the detection of enterovirus-71(EV-A71),coxsackievirus 6(CVA6),coxsackievirus 10(CVA10)and coxsackievirus 16(CVA16)using different cell generations were(8.20±0.07),(7.84±0.12),(7.93±0.09),(7.34±0.16)IgCCID/ml,respectively.The coefficient of variation(CV)ranged from 0.88%to 2.12%.Conclusions The established working cell bank of RD meets the requirements of quality control of Chinese Pharmacopoeia.The cells are stable,sterile and sensitive to enterovirus.It can be used for the determination of enterovirus vaccine-related viral titers and vaccine neutralizing antibody detection.

关 键 词：横纹肌肉瘤细胞 细胞库 传代稳定性 病毒敏感性 

分 类 号：R563.1[医药卫生—呼吸系统]