乳酸脱氢酶基因调控对HEK-293细胞代谢和腺病毒生产的影响

Effects of manipulating lactate dehydrogenase gene on metabolism of HEK-293 and production of human adenovirus

摘要:

减少乳酸积累一直是哺乳动物细胞生物技术产业的一个目标。体外培养动物细胞时，乳酸积累主要是2种代谢途径作用的综合结果：一方面，葡萄糖在乳酸脱氢酶A (lactate dehydrogenase A, LDHA)的作用下生成乳酸；另一方面，乳酸可通过乳酸脱氢酶B (LDHB)或乳酸脱氢酶C (LDHC)氧化为丙酮酸重新进入三羧酸循环。本研究综合评估了乳酸代谢关键基因调控对人胚胎肾细胞(human embryonic kidney 293 cells, HEK-293)细胞生长、代谢和人腺病毒(human adenovirus, HAdV)生产的影响，有效提高了HEK-293细胞的HAdV生产能力，并为哺乳动物细胞的乳酸代谢工程调控提供了理论基础。通过改造乳酸代谢关键调控基因(敲除ldha基因以及过表达ldhb和ldhc基因)，有效改善了HEK-293细胞的物质和能量代谢效率，显著提高了HAdV的生产。与对照细胞相比，3个基因改造均能促进细胞生长，降低乳酸和氨的积累，明显增强细胞的物质和能量代谢效率，显著提高了HEK-293细胞的HAdV生产能力。ldhc基因过表达对HEK-293细胞的生长、代谢和HAdV生产调控最显著，最大细胞密度提高了约38.7%，乳酸对葡萄糖得率和氨对谷氨酰胺得率分别下降了33.8%和63.3%，HAdV滴度提高了至少16倍。此外，相比于对照细胞株，改造细胞株的腺苷三磷酸(adenosine triphosphate, ATP)生成速率、ATP/O₂比率、ATP与腺苷二磷酸(adenosine diphosphate, ADP)的比值以及还原型辅酶Ⅰ (nicotinamide adenine dinucleotide, NADH)含量均有不同程度的提高，能量代谢效率明显改善。

Abstract:

Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured in vitro, the accumulation of lactate is mainly the combined result of two metabolic pathways. On one hand, glucose generates lactate under the function of lactate dehydrogenase A (LDHA); on the other hand, lactate can be oxidized to pyruvate by LDHB or LDHC and re-enter the TCA cycle. This study comprehensively evaluated the effects of LDH manipulation on the growth, metabolism and human adenovirus (HAdV) production of human embryonic kidney 293 (HEK-293) cells, providing a theoretical basis for engineering the lactate metabolism in mammalian cells. By knocking out ldha gene and overexpression of ldhb and ldhc genes, the metabolic efficiency of HEK-293 cells was effectively improved, and HAdV production was significantly increased. Compared with the control cell, LDH manipulation promoted cell growth, reduced the accumulation of lactate and ammonia, significantly enhanced the efficiency of substrate and energy metabolism of cells, and significantly increased the HAdV production capacity of HEK-293 cells. Among these LDH manipulation measures, ldhc gene overexpression performed the best, with the maximum cell density increased by about 38.7%. The yield of lactate to glucose and ammonia to glutamine decreased by 33.8% and 63.3%, respectively; and HAdV titer increased by at least 16 times. In addition, the ATP production rate, ATP/O₂ ratio, ATP/ADP ratio and NADH content of the modified cell lines were increased to varying degrees, and the energy metabolic efficiency was significantly improved.