葡萄糖水平对CEES染毒16HBE细胞能量代谢的影响及其机制  

Effect of glucose concentrations on energy metabolism in 16HBE cells exposed to CEES

作　　者：叶枫[1] 但国蓉[1] 王健[1] 赵远鹏[1] 赵吉清[1] 邹仲敏[1] 

机构地区：[1]第三军医大学军事预防医学院毒理学研究所,重庆400038

出　　处：《军事医学》2014年第3期169-173,共5页Military Medical Sciences

基　　金：全军医学科技"十二五"重大项目(AWS11C004);军队科技重大专项资助项目(2011ZXJ09102C)

摘　　要：目的：比较在高糖和低糖培养条件下，经半硫芥（CEES）染毒后支气管上皮细胞（16HBE）能量代谢的变化。方法采用高糖（4．5 mg/ml）和低糖（1．1 mg/ml）两种不同培养基培养16HBE细胞。 MTS法比较细胞增殖和CEES染毒后6 h细胞活性差异；用不同剂量CEES染毒两组细胞，24 h后HPLC检测胞内ATP、ADP、AMP含量，并计算ATP/ADP比值、腺苷酸池（总腺嘌呤核苷酸， total adenine nucleotides ， TAN）及能荷值（能量负荷值，energy charge， EC），Western印迹检测线粒体能量代谢功能酶COX-10和ISCU变化；流式细胞仪检测0．5 mmol/L CEES染毒后5、8、12、24 h时相点线粒体膜电位（MMP）变化。结果低糖培养的16HBE细胞的增殖明显增加，能量代谢指标ADP、TAN、COX-10和ISCU显著高于高糖组。高于0．5 mmol/L染毒剂量的CEES能显著降低高糖、低糖培养的16HBE细胞的活性，且在1．0 mmol/L下两组间差异有显著意义。高糖组在0．5、1．0 mmol/L染毒24 h后，ATP、ADP、TAN显著增高，而ATP/ADP比值及EC显著降低。低糖组在1．0 mmol/L染毒24 h后，ADP、AMP、TAN显著低于染毒前，而ATP/ADP比值及EC显著高于染毒前。在0．5 mmol/L CEES染毒下，高糖组线粒体膜电位在8～12 h显著增加，其后至24 h时恢复至正常水平。低糖组MMP在5 h有一过性显著降低，8 h后与染毒前已无显著差异。高糖培养时，16HBE细胞的氧化磷酸化相关蛋白COX-10和ISCU蛋白水平显著低于低糖培养的细胞；0．5～1．0 mmol/L CEES染毒24 h后，两者的水平显著升高，与低糖组已无显著差异。1．0 mmol/L CEES染毒24 h后，低糖组COX-10和ISCU水平显著降低。结论糖浓度差异会影响16 HBE细胞的能量代谢、增殖和CEES损伤后能量应激反应。高糖可能通过增加细胞的应激反应能力抵抗CEES的细胞毒性作用。Objective To compare the changes in energy metabolism in 2-chloroethyl ethryl sulfide（CEES）-poisoned bronchial epithelial cell 16HBE cultured in media at different glucose concentrations .Methods Bronchial epithelial cell 16HBE was cultured in high （4.5 mg/ml） or low （1.1 mg/ml） glucose medium and exposed to a sulfur mustard simulant CEES of 0.2, 0.5, 1.0 mmol/L.Cell growth and cytotoxicity were tested using MTS .ATP, ADP and AMP were detected by HPLC and the value of ATP/ADP, total adenine nucleotides （ TAN） and energy charge （ EC） was subsequently calculat-ed.Mitochondrial oxidative phosphorylation-related proteins, COX-10 and ISCU, were detected using Western blotting . Rhodamine 123 was applied to detect the mitochondrial membrane potential using flow cytometry .Results Low glucose accelerated the growth and energy metabolism of 16HBE cells in regular culture , and the contens of ADP , TAN, COX-10 and ISCU in low glucose group were significantly higher than those in high glucose group .CEES exposure （≥0.5 mmol/L） significantly affected cell viability in both high and low glucose groups , with significant difference between the two groups exposed to 1.0 mmol/L CEES.In high glucose group, 24 h after 0.5 or 1.0 mmol/L CEES exposure, the contents of ATP, ADP and TAN were significantly increased , while ATP/ADP and EC decreased .In low glucose group , ADP, AMP and TAN significantly decreased, while ATP/ADP and EC increased 24 h after 1.0 mmol/L CEES exposure.The mi-tochondrial membrane potential （MMP） also changed differently after 0.5 mmol/L CEES exposure.MMP in high glucose＆amp;nbsp;group marginally increased at 3 h, and significantly increased at 8-12 h （P＆lt;0.05）, and returned to normal at 24 h. MMP in low glucose group showed a transient decrease at 5 h （P＆lt;0.01）, and back to normal at 8 h.The protein levels of COX-10 and ISCU were significantly increased in high glucose group 24 h after 0.5-1.0 mmol/L CEES exposure , but sig-nificantly decreased in low one 24 h

关 键 词：葡萄糖 半硫芥 能量代谢 线粒体 

分 类 号：R827.1[医药卫生—临床医学]