探討葡萄糖濃度對中國大倉鼠卵巢細胞增生之研究

Abstract

本實驗室先前研究發現CHO細胞株在高葡萄糖濃度（10 g/L）下，葡萄糖消耗速率會增加2倍，乳酸累積速率增加30 ﹪，而生長倍增時間由13小時延長為18小時，並且細胞內cAMP含量增加2∼3倍。在人類血管內皮細胞亦發現在高葡萄糖濃度下，細胞倍增時間與細胞內cAMP皆有增加之趨勢。由於細胞內之cAMP為二級訊息傳導物，對細胞內酵素活性、細胞分化及增生等均扮演重要角色。由體外培養之肝細胞及人類乳癌細胞中發現，當提高細胞內cAMP含量，會調控MAP kinase磷酸化，進而抑制細胞增生，且MAP kinase活性和細胞分化及增生亦有關。 為了解此高葡萄糖濃度抑制是否受不同濃度血清之影響，我們利用低血清（2 %）培養CHO細胞株，來觀察高及低（1 g/L）濃度葡萄糖在低血清濃度下對其生理影響，結果我們發現在高葡萄糖濃度環境下，細胞對葡萄糖消耗速率增加2倍、乳酸累積速率增加50 ﹪、細胞內cAMP含量增加約3倍，此結果和高血清（10 %）培養條件下有相近結果，因此cAMP變化主要受葡萄糖濃度影響。由於在高葡萄糖濃度下，我們發現MAP kinase活性有上升現象，但加入PKA(cAMP dependent Protein kinase A) 抑制劑(H-8)後，MAP kinase活性則與低葡萄糖濃度相同；此外，亦觀察不同細胞週期下葡萄糖對cAMP之影響，結果發現高葡萄糖濃度下，細胞內cAMP含量皆高於低葡萄糖濃所培養之細胞，且在G1時期表現最高，而MAP kinase活性在高葡萄糖濃度下則有延長現象。綜合以上實驗結果我們推論，葡萄糖可能經由cAMP活化MAP kinase或抑制 dephosphorylation，繼而延長細胞增生時間。進一步實驗證實在加入PKA抑制劑(H-8)後，高葡萄糖濃度之細胞生長速率回復至1.3/day，此結果與低葡萄糖濃度之細胞生長速率一致，因此本篇論文推論高葡萄糖濃度對細胞生長速率之抑制是經由cAMP調控，進而活化MAP kinase所影響。

The high glucose concentration inhibits Chinese Hamster Ovary (CHO) cell growth but the mechanism is still unclear. In previous studies, we found that the cell growth rate decreased from 1.3/day to 0.9/day when cells were grew in 10 g/L glucose concentration. In addition, the glucose consumption rate, lactate production rate, and the intracellular cAMP level increased twofold, 30 %, and threefold respectively. In this study, we have further demonstrated that the inhibition of the CHO cell growth was directly affected by high glucose concentration because it happened at both of high and low serum concentrations medium. The intracellular cAMP level regulates cell proliferation at high glucose growth condition in fibroblast cells. Therefore, the increase of cAMP might mediate the activation of Mitogen-activated Protein (MAP) kinase activity to inhibition of the CHO cells’ growth. In this study, the results showed that the MAP kinase phosphorylation did increase at high glucose concentration condition, and this activation was returned to the same level as grew in low glucose concentration when the cAMP signal was blocked. The high glucose concentration also regulated the CHO cell’s cell cycle during the G1 phase (0 to 8 hr growth). The intracellular cAMP level was increased and the phosphorylation of MAP kinase was postponed (or the dephosphorylation activity was inhibited) when glucose concentration was increased from 1 g/L to 10 g/L. Interestingly, the intracellular cAMP signals were blocked by cAMP dependent protein kinase A (PKA) inhibitor, and the MAP kinase phosphorylation activity as well as cell growth rate were returned to the same level as in low glucose concentration. Therefore, the results of these studies suggested that the high glucose might activate the MAP kinase activity by elevation of the intracellular cAMP level to inhibit the CHO cell growth.