重组CHO-GS细胞降低氨毒副作用的代谢研究  

Metabolism of Recombinant CHO-GS Cell Reducing of Toxic Effect of Ammonia

作　　者：张芳[1] 易小萍[1] 孙祥明[1] 张元兴[1] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《生物工程学报》2006年第1期94-100,共7页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划资助项目(863计划)(No.2004AA2Z3792)~~

摘　　要：在重组CHOGS细胞无血清批培养过程中,由于GS系统的引入,使氨对细胞的毒副作用显著降低,从而引起细胞生长和代谢途径发生变化。当起始氨浓度为1.42mmolL时,细胞最高密度可达到15.6×105cellsmL,随着氨浓度的增加,尽管细胞生长受到一定的抑制,但在氨浓度为12.65mmolL时,细胞密度仍可达到8.9×105cellsmL。当起始氨浓度从0.36mmolL增加到12.65mmolL时,细胞对葡萄糖的得率系数和乳酸对葡萄糖的得率系数降低,己糖激酶(HK)、丙酮酸激酶(PK)和乳酸脱氢酶(LDH)酶活分别提高了43%、140%和25%,表明细胞对葡萄糖的利用增加,糖代谢更倾向于高能量生成途径。在谷氨酰胺代谢途径中,氨促进了谷丙转氨酶(GPT)酶活,谷氨酸到α酮戊二酸的转化逐渐倾向于谷丙转氨途径,谷氨酸脱氢酶(GDH)酶活降低,脱氨途径相应受到抑制。此外,氨浓度的增加使细胞群体处于G0G1期的比例逐渐升高,当氨浓度为12.65mmolL时,重组蛋白比生产速率比氨浓度为0.36mmolL时提高了2.1倍。The toxic effect of ammonia on rCHO-GS cell decreased obviously due to the transfection of GS system in serum-free culture. The maximum cell density, 15.6 × 10^5 cells/mL was obtained in the culture with 1.42 mmol/L ammonia. The growth of rCHO-GS cell was inhibited with an increased ammonia concentration. However, a cell density of 8.9 × 10^5 cells/mL was obtained when the concentration of ammonia was 12.65mmol/L. The intracellar metabolic pathways were affected due to the decrease of the toxic effect of ammonia on rCHO-GS cell. With the increase of initial ammonia concentration from 0.36mmol/L to 12.65mmol/L, the yield coefficients of cell to glucose and lactate to glucose decreased. The activities of hexokinase （HK）, pyruvate kinase （PK）, and lactate dehydrogenase （LDH） increased by 43%, 140% and 25%, respectively, indicating that the utilization of glucose increased and the glycolysis pathway was more prone to efficient energy metabolism pathway. An increased activity of glutamate-pyruvate aminotransferase （GPT） showed that the conversation from glutamate to α-ketoglutarate was shifted to glutamate-pyruvate transamination pathway. The deamination pathway was inhibited due to a decreased activity of glutamate dehydrogenase. In addition, the number of cells in G0/G1 phase increased and the specific production rate of recombinant protein increased by 2.1-fold with the increase of initial ammonia concentration from 0.36mmol/L to 12.65mmol/L.

关 键 词：重组CHO细胞 谷氨酰胺合成酶 无血清培养 代谢 氨 

分 类 号：Q813.11[生物学—生物工程]