生产禽流感病毒的悬浮MDCK细胞稳定性的研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-0434

摘要/Abstract

摘要：

旨为研究悬浮MDCK细胞在长期传代过程中细胞生长稳定性与病毒生产稳定性。以悬浮MDCK细胞为研究对象,通过对该细胞进行了长达200多天的传代培养,考察了传代过程中各代次细胞的生长情况以及对H9N2禽流感病毒的生产能力差异;探究了不同培养基稀释比例和MOI等工艺参数条件对长期传代的悬浮MDCK细胞生产H9N2禽流感病毒过程的影响关系;另外,从ROS水平和细胞周期两方面,研究了高低代次细胞的病毒生产能力差异的内在原因。结果显示,各代次悬浮MDCK细胞形态相似,细胞生长能力无显著性差异,表明该细胞在长期传代过程中细胞生长稳定性良好。但HA滴度随细胞代次的增加而增加,最高代次细胞（P111）的病毒滴度较最低代次细胞（P1）约高0.5 Log₂HAU/100 μL;P111细胞在不同工艺下病毒产量均高于P1细胞,表明了该细胞长期传代后病毒生产能力略有提高。最后,研究发现,与P1相比,P111胞内ROS水平较低并且G0/G1期细胞的比例较高,此现象可能是高代次细胞产量提高的原因。

关键词: 悬浮MDCK细胞, 禽流感病毒, 细胞株稳定性, ROS, 细胞周期

Abstract:

The aim of this study is to determine the stability of growing and virus producing for suspension MDCK cells during long-term passaging culture. Suspended MDCK cells were subcultured for more than 200 d. Taking suspended MDCK cells at different passaging time points,their growth patterns and the difference in virus production of H9N2 avian influenza virus（H9N2-AIV）were investigated. The effects of different process parameters and conditions such as medium dilution ratios and MOI on the production of H9N2-AIV from the suspended long-term passaging MDCK cells was explored. The reasons for the difference in H9N2-AIV production capacity between the lowest passaged cells（P1）and the highest passaged cells（P111）were preliminarily investigated from two aspects,i.e. the intracellular ROS level and cell cycle. The results showed that the MDCK cells at the different passages presented similar morphology,and insignificant difference was observed concerning cell growth between all subcultures and batch cultures,which indicated that the growth of the MDCK cells were stable during long-term passaging. The HA titer increased with elevated cell passaging time,the HA titer of P111 was about 0.5 Log₂HAU/100 μL higher than that of P1. Meanwhile,P111 cells produced higher virus than P1 cells under different tested processes,indicating that the H9N2-AIV production capacity of the suspended MDCK cells had slightly increased with the increase of cell passaging time. It was also found that P111 cells had lower ROS level and higher proportion of G0/G1-phase cells,which may be the main reasons for the increased yield of H9N2-AIV from MDCK cells.

Key words: suspended MDCK cells, avian influenza virus, cell line stability, ROS, cell cycle

白春丽, 叶倩, 姬阿美, 刘旭平, 张旭, 刘志亮, 朱明龙, 赵亮, 谭文松. 生产禽流感病毒的悬浮MDCK细胞稳定性的研究[J]. 生物技术通报, 2020, 36(10): 72-79.

BAI Chun-li, YE Qian, JI A-mei, LIU Xu-ping, ZHANG Xu, LIU Zhi-liang, ZHU Ming-long, ZHAO Liang, TAN Wen-song. Stability of Suspended MDCK Cells for Avian Influenza Virus Production[J]. Biotechnology Bulletin, 2020, 36(10): 72-79.

图/表 8

图1 各代次悬浮MDCK细胞的形态

图2 各代次悬浮MDCK细胞传代过程中活细胞密度（A）,细胞直径（B）以及比生长速率（C）变化

图3 各代次悬浮MDCK细胞批培养过程中活细胞密度（A）,细胞活率（B）以及比生长速率（C）变化

图4 各代次悬浮MDCK细胞流感病毒HA滴度（A）与单细胞产毒率（B）随时间变化

图5 不同稀释比例下高低代次悬浮MDCK细胞所产流感病毒最高HA滴度（A）与最高单细胞产毒率（B）

图6 不同MOI下悬浮MDCK细胞生产流感病毒HA滴度与单细胞产毒率随感染时间的变化

图7 产毒期P1与P111的胞内ROS水平

表1 病毒感染前P1与P111的细胞周期分布

Passage	细胞周期分布/%
	G0/G1	S	G2/M
P1	46.16±0.67	24.18±0.43	26.44±1.31
P111	57.58±0.36	19.36±0.29	20.58±0.33

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