动物细胞培养过程中活性氧对乳酸代谢特征转变的影响及调控机制

The adverse effects of lactate accumulation on antibody productivity of mammalian cells were well-known. A metabolic shift from lactate production to net lactate consumption phenotype was desirable and identified as a critical process parameter correlated to high productivity. However, this metabolic shift was neither generic nor could it be hardly controlled, as the mechanism modulating lactate production/consumption in cell culture was still under investigation. In our previous studies, a correlation between reactive oxygen species (ROS) and lactate-shift was observed. To further investigate lactate-shift mechanism, several engineered Chinese hamster ovary cell lines (CHO lines) producing “blockbuster” therapeutic antibodies will be employed in this study. Lactate profiles will be first observed during different ROS levels. And the speculation that lactate shift is ascribed to ROS accumulation will be verified. Then, the main signal pathway and key steps of lactate-shift will be clarified by analyzing the performance of p53 expression, phosphofructokinase activity, cytochrome c oxidase activity, pyruvate concentration, NADH concentration, glycolytic pathway level, oxidative phosphorylation level, and lactate response when up-regulating/down-regulating ROS level. And the mechanism of lactate-shift caused by ROS will be further understood. On this basis, lactate-shift manual regulation strategy will be proposed with aim to reduce lactate concentration during the antibody production stage. A high-yielding process will be further estabilished with the increased cell density, improved antibody productivity, and prolonged culture time. This research will facilitate the high-yielding manufacture of therapeutic antibodies by mammalian cell culture directly, and provide new perspectives for development of “super”-CHO cell lines.

本项目将紧紧围绕动物细胞培养过程优化面临的乳酸代谢难以人为调控这一关键技术难题，以表达具有广阔市场前景的抗体蛋白药物的CHO工程细胞为研究对象，基于代谢工程和过程优化的基本理论和方法，从减少乳酸累积并促进其消耗、提高营养物利用率出发，首先认识胞内活性氧水平与乳酸生成和消耗的关系，揭示其对乳酸代谢特征转变所发挥的重要作用，随后通过分子水平、蛋白质水平以及代谢网路的研究和分析，认识活性氧水平对p53蛋白表达、磷酸果糖激酶和细胞色素氧化酶酶活、胞浆丙酮酸和NADH浓度、糖酵解途径流量和氧化磷酸化水平等与乳酸代谢相关的上下游途径和节点的影响，以进一步揭示工程细胞在培养过程中发生乳酸代谢特征转变的作用机制，从而掌握有效调控乳酸代谢行为的科学方法，为未来优化工业规模动物细胞培养过程、提高抗体等重组蛋白药物的生产效率提供科学指导。此外，本项目研究成果也将为构建更为高效高产的工程细胞株提供新的思路。

本项目围绕动物细胞培养过程优化面临的乳酸代谢难以人为调控这一关键技术难题，以表达抗体蛋白药物的CHO工程细胞为研究对象，首先分析了不同乳酸代谢特征下胞内氧化状态的差异，结果显示，处于乳酸消耗状态下的细胞伴随着更高的氧化状态，细胞氧化还原电势从-705.45±12.67 mV提高到-363.78±13.98 mV）。其次，采用MFA分析手段对两个典型乳酸代谢培养过程进行分析，结果显示，提高胞内氧化状态后（即乳酸代谢转变），糖酵解速率降低了37.12%，丙酮酸到乙酰辅酶A的代谢通量增加了13.33%，三羧酸循环通量提高了23.19%，胞浆中NAD+/NADH较乳酸生成期提高1.48倍。这些代谢的变化导致胞浆中丙酮酸浓度降低。胞内氧化能力的提高（NAD+/NADH）以及胞浆丙酮酸浓度的下降为乳酸向丙酮酸转化提供了驱动力。最后采用分子生物学的手段分析胞内氧化状态、p53蛋白和乳酸代谢三者之间关系，发现提高胞内氧化状态后，活性氧水平提高了30-40%，为了避免由高浓度活性氧引起氧化应激损伤，p53蛋白的表达被激活（其mRNA表达量提高了84.21%）。p53蛋白表达水平的提高一方面抑制下游PFK-1的表达水平（降低29%）和活性（降低25%），降低了糖酵解速率，另一方面细胞色素C氧化酶-2（SCO2）mRNA相对表达量提高了42.85%，增强了氧化磷酸化水平。综上所述，乳酸代谢转变调控机制可描述为ROS的累积提高胞内氧化状态，细胞为了维持氧化还原状态平衡，激活了抗氧化剂p53蛋白的表达，p53蛋白作为关键调控因子抑制糖酵解途径，增强TCA循环和氧化磷酸化水平，导致胞浆中NADH和丙酮酸浓度降低，为乳酸向丙酮酸转化的提供驱动力。通过本项目的研究，深入认识了胞内氧化状态在CHO细胞乳酸代谢转变中所发挥的作用，可望对细胞培养过程中乳酸代谢转变进行有效调控，为解决细胞培养过程中乳酸积累问题提供借鉴。