葡萄糖濃度對中國大倉鼠卵巢細胞生長速率、能量狀態與代謝影響之研究

Abstract

中國大倉鼠卵巢細胞（Chinese hamster ovary cell；CHO）是工業上廣泛用來生產醫療用蛋白質之哺乳動物細胞株。而哺乳動物細胞之生長、代謝與生產目標蛋白質效率會受到培養基中葡萄糖濃度的影響，因此本篇論文針對CHO細胞在不同葡萄糖濃度的培養下，其細胞生理、生長與能量代謝等各方面進行研究。 實驗結果顯示葡萄糖濃度改變會影響CHO-K1細胞生長速率，當葡萄糖濃度由1 g/L提高至10 g/L時，細胞生長速率1.3 d-1下降為0.9 d-1，其倍增時間(doubling time)由原來之13小時延長為18小時。葡萄糖消耗速率則由1.3 mg/d/106cells升高兩倍成2.7 mg/d/106cells，而乳酸累積速率也上升了43 %，氨的累積速率則無顯著的差別。研究不同葡萄糖濃度對於CHO-K 1能量狀態的影響，發現細胞內ATP含量由6.1 nmol/106 cells略升高至7.0 nmol/106 cells，而ATP/ADP比例則上升了40 %。再者，當生長速率為1.3 d-1時，細胞內cAMP含量為365 fmol/106 cells，當生長速率為下降為0.9 d-1時，細胞內之cAMP含量上升4.5倍成為1650 fmol/106 cells。研究葡萄糖代謝酵素活性時，我們發現hexokinase (HK)與其它酵素比較，其比活性為最低，因此推測HK為CHO-K1細胞進行糖解作用時之速率決定步驟酵素。而提高葡萄糖濃度至10 g/L培養時，檸檬酸循環中isocitrate dehydrogenase (ICDH) 比活性下降了50 %，所以ICDH對於調節檸檬酸循環之活性可能也扮演著重要角色。

A variety of Chinese hamster ovary (CHO) cell lines are widely used in the production of therapeutically important proteins. Glucose metabolism in some mammalian cell lines has been investigated on correlation of specific utilization and production rates. In this study, we studied the effect of glucose concentrations on CHO-K1 cell growth, energy state and metabolism. The results showed that the growth rate of Chinese hamster ovary cells was inhibited by high glucose concentration. The cell specific growth rate decreased from 1.3/day to 0.9/day while glucose concentration increased from 1 g/L to 10 g/L. The glucose consumption rate and lactate production rate increased two folds and 40% respectively under high glucose concentration, whereas ammonium production rate was remained constantly. In addition, the intracellular ATP concentration was slightly increased, and ATP/ADP ratio was elevated 40% at high glucose concentration. The intracellular cyclic AMP concentration was 4.5 folds at high glucose concentration of 10 g/L. Hexokinase was the rate-limiting enzyme of glycolytic enzymes and the specific activity of isocitrate dehydrogenase decreased 50% at high glucose concentration. These results demonstrated that CHO cells metabolism and cell growth are controlled by different glucose concentrations in the medium.