高细胞密度下流感病毒引起的MDCK细胞凋亡机制研究

Due to increasing requirements for vaccine production, large amounts of infectious viral particles need to be generated by designing efficient and scalable virus production processes. However, one of the major restrictions on the production of virus is the cell density effect: there is a drop in cell-specific virus productivity associated with increased cell concentration at infection, and the production of virus is limited by low cell densities. The Cell density effect is caused by several factors. Our researchs show that the most important factor maybe is the apoptosis of the cell at the time of infection which is probably a determining factor for virus production. Therefore, in this study, the mechanism of generation of cell apoptosis in high cell density culture and its infection of influenza virus replication process will be study in MDCK cells. Through focus on the effect of virus infection parameters, culture environment and operating conditions on cell apoptosis, we will understand the variation of the proportion of apoptosis in high cell density conditions and reveal its mechanism of cell apoptosis effect on virus production process. By analyzing the activities of key enzymes and proteins of different cell apoptotic pathways, we will understand the mechanism of cell apoptosis in different cell density culture, and clarify the main pathway and the key steps of cell apoptosis. On this basis, further analysis of the influence of cell apoptosis on virus infection and replication process will be done to find out the key limiting factor in virus production. After that, we take appropriate measures to improve the efficiency of virus production, and thus cost-effective vaccines industrial production process could be establish. At the same time, this study will promote the vaccine industry based on large-scale production of cells and improve the efficiency of virus production.

在细胞生长到高密度进行感染病毒时，单位细胞病毒扩增效率下降导致病毒产量低，已成为限制基于细胞培养生产疫苗高效工业化生产的关键性难题之一。前期研究表明，病毒引起的细胞凋亡可能是显著影响因素之一。本项目将在前期工作基础上，以流感病毒和MDCK细胞为研究体系，紧紧围绕高细胞密度条件下病毒扩增效率低这一核心问题，通过研究病毒感染参数、培养环境、操作条件对细胞凋亡的影响，认识高细胞密度条件下细胞凋亡比例变化的规律，揭示其对病毒感染复制过程的影响，并在此基础上分析细胞凋亡途径的关键酶活和蛋白表达，进一步阐明细胞凋亡的机制，为提高病毒产量，实现疫苗产能的突破，建立经济有效的疫苗工业化生产过程奠定基础。同时，本项目的研究结果对基于细胞生产疫苗行业的规模化生产有重要的指导意义。

在细胞生长到高密度进行感染病毒时，单位细胞病毒扩增效率下降导致病毒产量低，已成为限制基于细胞培养生产疫苗高效工业化生产的关键性难题之一。本项目在前期工作基础上，以流感病毒和MDCK细胞为研究体系，紧紧围绕高细胞密度条件下病毒扩增效率低这一核心问题，通过研究病毒感染参数、培养环境、操作条件对细胞凋亡的影响，认识了高细胞密度条件下细胞凋亡比例变化的规律，揭示了其对病毒感染复制过程的影响，为提高病毒产量，实现疫苗产能的突破，建立经济有效的疫苗工业化生产过程奠定基础。首先从细胞生长和病毒增殖效率两方面考察各种影响因素，确立了基于MDCK细胞培养的流感疫苗高效生产工艺。首先考察了TPCK-胰酶浓度、MOI和TOI三个感染参数。最适TPCK-胰酶浓度和MOI分别为5.0 mg/L和0.01；单位细胞病毒产率Svy随TOI增大而降低，TOI为72 h时病毒滴度最高而Svy低下，表明产率仍有较大提升空间。因此，为进一步提高该TOI条件下的Svy，考察了营养和培养环境条件对病毒扩增的影响。通过换液培养证明可通过调整产毒期营养提高Svy；以基础营养物优化后的培养基扩增病毒，HA滴度和Svy均显著提高；产毒期添加丁酸钠亦能显著提高Svy。DO对病毒产率无显著影响；pH可显著影响病毒产率，最适细胞生长期pH（pHg）为7.0，最适产毒期pH（pHpi）为7.2；温度亦显著影响病毒产率，细胞生长期温度（Tg）和产毒期温度（Tpi）最适值均为35°C。据此，确立了基于MDCK细胞培养的流感病毒高效生产工艺，HA滴度达213 HA units/50 µL，Svy达（41.31±0.98）×103 virions/cell。综上所述，pHpi、Tg和Tpi等培养环境条件对MDCK细胞中流感病毒增殖效率的影响机理为：控制适当pHpi、Tg和Tpi均可有效调控细胞生理状态（主要表现为提高G0/G1期细胞比例和减少凋亡发生），使之更利于病毒感染和进入细胞，且延长病毒在胞内的扩增时间。同时，在此种细胞生理状态下，病毒RNA酶表达量增加，且胞内抑病毒蛋白EBP1表达量下降，使病毒RNA复制效率得到有效提高。通过本文的研究，开发了基于MDCK细胞培养的流感病毒疫苗高效生产工艺，为流感疫苗的工业化生产奠定了基础。