悬浮与贴壁MDCK细胞在流感病毒分离培养中的应用评价  

Comparison of isolating influenza virus in culture systems of adherent and a suspending cells of MDCK cell line

作　　者：周雪 俞俊岭 杨思天 夏艺丹 罗婉蓉 方惟希 胡敏昊 何军[1] Zhou Xue;Yu Junling;Yang Sitian;Xia Yidan;Luo Wanrong;Fang Weixi;Hu Minhao;He Jun(Anhui Provincial Center for Disease Control and Prevention(Public Health Research Institute of Anhui Province),Hefei 230601,China;College of Public Health,Anhui Medical University,Hefei 230000,China)

机构地区：[1]安徽省疾病预防控制中心(安徽省公共卫生研究院),合肥230601 [2]安徽医科大学公共卫生学院,合肥230000

出　　处：《中华实验和临床病毒学杂志》2023年第6期637-642,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：2021年度安徽省卫生健康科研项目(AHWJ2021a030);科技部国家重点研发计划(2022YFE0110100);2022年安徽省新冠病毒科研应急攻关项目(2022e07020071)。

摘　　要：目的比较悬浮细胞MDCK和贴壁细胞MDCK两种培养体系对流感病毒分离培养的差异,探讨悬浮细胞的应用前景。方法通过细胞计数记录两种细胞的活细胞密度和细胞特定生长速率,使用WHO推荐疫苗株进行病毒感染实验,连续传代5次后观察血凝效价并对HA和NA基因进行测序,观察基因突变情况。结果悬浮细胞的24 h、48 h活细胞密度较贴壁细胞更稳定;病毒连续传代培养至第三代时悬浮细胞培养的病毒HA滴度可达1∶256以上,而贴壁细胞则无滴度;悬浮细胞培养的H3N2和BV亚型流感病毒第四代和第五代HA基因各发生1个氨基酸位点突变,贴壁细胞两个代次内未发现基因突变,NA基因均未发现基因突变。结论悬浮MDCK细胞较贴壁细胞生长更为稳定,且病毒分离培养效率高,连续传代病毒突变率低,在基于细胞培养技术的流感疫苗生产工艺中具备一定的可行性。Objective To compare the differences between suspension and adherent cells of MDCK cell line in the isolation of influenza virus,and to explore the application prospects of MDCK cell suspension.Methods Determination of viable cell density and cell specific growth rate were recorded by cell count.The WHO recommended vaccine strains were used for virus infection experiments.After five passages,hemagglutination titers were detected,while the sequencing analysis of their HA and NA genes revealed the mutation frequency.Results The 24-hour and 48-hour viable cell density of the cell suspension was more stable than that of adherent cells.The cell suspension achieved an HA titer of 1∶256 or higher in the third generation,while adherent cells had no titer.In the fourth and fifth generations,one amino acid site mutation was found in the HA gene of H3N2 and BV subtypes of influenza virus cultured in the cell suspension,while no gene mutation was found in adherent cells in two passages.There were no mutations in the whole NA gene.Conclusions Suspension of MDCK cells have more stable growth and higher efficiency in virus isolation than adherent cells,meanwhile there was a low rate of virus mutation during continuous passage.This study demonstrated the feasibility of this suspension of MDCK cells for influenza vaccine production based on cell culture technology.

关 键 词：流感病毒 悬浮MDCK细胞 贴壁MDCK细胞 病毒分离 

分 类 号：R373[医药卫生—病原生物学]