采用代谢流量分析方法评估二噁英对细胞的代谢干扰  

The influence of dioxin on cell metabolism evaluated by the metabolic network flexibility analysis

作　　者：张保琴[1] 张海军[1] 王龙星[1] 王金成[1] 杨常青 陈吉平[1] 

机构地区：[1]中国科学院大连化学物理研究所,大连116023 [2]满洲里出入境检验检疫局技术中心,满洲里021400

出　　处：《环境化学》2012年第11期1797-1802,共6页Environmental Chemistry

基　　金：国家重点基础研究发展计划(2009CB421602)资助

摘　　要：以肝癌细胞HepG2为供试细胞,选用5个不同浓度的二噁英(0、0.001、0.01、0.1和1.0 nmol.L-1),探讨2,3,7,8-TCDD对HepG2细胞的毒性效应.采用90%甲醇离心法实现蛋白与代谢产物的分离,建立了高效液相色谱-串联三重四极杆质谱(HPLC-MS/MS)分析胞外液中的23种代谢产物(20种氨基酸、乳酸、甘油和尿素)的方法.建立的HPLC-MS/MS分析方法对各化合物的加标回收率可达91%—105%之间,化合物浓度的标准偏差均小于10%.在此基础上,结合代谢流量分析系统研究了2,3,7,8-TCDD对HepG2细胞内代谢流的影响.结果表明,随着浓度的增高,2,3,7,8-TCDD对HepG2细胞代谢的干扰作用呈增强趋势.2,3,7,8-TCDD降低了细胞对葡萄糖的吸收,并进一步抑制了HepG2细胞糖酵解的速率.此外,2,3,7,8-TCDD导致了HepG2细胞乳酸代谢流的明显增加,由此使细胞产生了过量的乳酸.This study was initiated to explore the toxic effects of dioxins in HepG2 cells when exposed to 2,3,7,8-TCDD at five different concentrations （0, 0. 001, 0.01, 0.1, 1.0 nmol- L-1 ）. The separation of proteins and metabolites was achieved by the centrifugation method with 90% methanol as solvent, and then 23 extracellular metabolites （20 underivatized amino acids, lactate, glycerol and urea） were qualitatively and quantitatively analyzed by HPLC-MS/MS. The recoveries of the 23 cell metabolites were 91%--105% and the relative standard deviations （RSDs） of peak areas of the extracted ion chromatograms were below 10%. Meanwhile, the influence of 2,3,7,8-TCDD on the metabolic flux analysis in HepG2 cells was evaluated. The results showed that the disturbance of 2,3,7,8-TCDD on the metabolism of HepG2 increased its concentration. The addition of 2,3,7,8-TCDD decreased the cell absorption of glucose and further inhibited the glyeolysis. On the other hand, exposure to 2,3,7,8-TCDD induced a significant increase of lactic acid metabolic flux, and thus caused excessive lactic acid in HepG2 cells.

关 键 词：HPLC-MS MS 代谢流量分析 细胞代谢产物 2 3 7 8-TCDD 

分 类 号：TQ922.9[轻工技术与工程—发酵工程]