摘要
目的了解人表皮角质细胞培养过程中,氨对细胞生长和代谢的影响。方法收集3～6岁健康幼儿包皮,以DispaseⅡ分离、收集原代细胞,角质化细胞专用无血清培养基(Keratinocyte-SFM)培养。待细胞培养至第4代,更换含不同氨浓度的培养基,考察细胞生长和代谢的变化。结果氨抑制了细胞的生长,当氨的浓度为9.23mmol/L时,脉冲培养结束时的活细胞密度(0.84×105个/ml)进一步下降,细胞存活率下降至54.5%;氨对细胞生长的抑制作用遵循二级抑制模型,其模型常数Ka为4.46(mmol/L)2,即当氨的浓度为2.11mmol/L时,细胞的比生长速率下降到最大比生长速率的50%。当氨浓度从1.23mmol/L增加到1.73mmol/L,细胞对葡萄糖的得率系数下降了48.0%,葡萄糖的比消耗速率上升了14.8%;与氨浓度为1.23mmol/L相比,9.23mmol/L的氨使乳酸对葡萄糖的得率系数增加了12.0%,乳酸的比生成速率增加了53.3%。氨浓度的增加明显改变了细胞的代谢,促进了细胞对葡萄糖的利用,消耗的葡萄糖更倾向于乳酸的生成。当氨浓度为9.23mmol/L,谷氨酰胺的比消耗速率和氨的比生成速率分别是氨浓度为1.23mmol/L的20.2%和6.2%。氨抑制了谷氨酰胺的消耗,谷氨酰胺在生成谷氨酸后经过脱氢途径的流量减少,更多地向转氨途径,即低效率的产能途径偏移。结论在人表皮角质细胞的培养过程中,应尽量减少氨的积累。
Objective To investigate the influence of ammonia in serum-free medium on growth and metabolism of human keratinocytes. Methods Primary human keratinocytes were isolated from children foreskin by two-step digestion using trypsin and dispase II. Ammonia at various concentrations was added into the human keratinocytes, and the growth and metabolism of the cells were observed. Results The cell growth was inhibited by ammonia obviously. The viable cell density and cell viability were reduced to 0.84×105 cells/ml and 54.5% respectively by using ammonia with an initial concentration of 9.23 mmol/L. The inhibition of ammonia on the growth of cells was consistent with the second-order inhibition model, and the inhibition constant Ka is 4.46 (mmol/L)2, i.e. the specific growth rate of the cells was reduced to 50% of its maximum value by using 2.11 mmol/L ammonia. The metabolic pathways were altered in the cultures with ammonia concentrations. With an increase of the ammonia concentration from 1.23 to 1.73 mmol/L, the yield coefficients of cell growth to glucose consumption decreased by about 48%, and the specific rate of glucose consumption increased by 14.8% . Compared to the control group, the yield coefficients of lactate production to glucose consumption and specific rate of lactate production increased by 12.0% and 53.3%, respectively by using 9.23 mmol/L ammonia. The glucose consumption was enhanced and was more prone to produce lactate by anaerobic metabolism. When ammonia concentration was 9.23 mmol/L,specific rates of glutamine consumption and ammonia production were 20.2% and 6.2% respectively in the control group. Ammonia reduced glutamine consumption as a whole. The reaction of glutamate to α-ketoglutarate stimulated by glutamate dehydrogenase was inhibited by ammonia, and that stimulated by alanine aminotransferase was facilitated, which made glutamine produce more energy in a low efficient metabolic pathway. Conclusions It is necessary to reduce ammonia accumulation in keratinocytes culture.
出处
《中国医药生物技术》
CSCD
2007年第2期123-129,共7页
Chinese Medicinal Biotechnology
关键词
氨
细胞生长过程
代谢
角蛋白细胞
Ammonia
Cell growth processes
Metabolism
Keratinocytes
作者简介
崔磊,Email:cuileite@yahoo.com.cn 通讯作者
