摘要目的 探讨间歇性低氧对肝细胞脂质代谢的影响及其机制.方法 利用三气培养箱(O2、CO2、N2体积分数分别为2％、5％、93％)8 h/d间歇性低氧处理L02和HepG2细胞1、2、3、4、5d,建立间歇性低氧细胞模型,油红O染色及甘油三酯(TG)测定检测肝细胞内的脂滴含量,流式细胞仪及荧光显微镜观察间歇性低氧对肝细胞内活性氧(ROS)的影响,Western blot检测ROS对低氧诱导因子(HIF)-1α和HIF-2 α蛋白表达的调节,Western blot检测HIF-1α下游与脂质代谢相关的固醇调节元件结合蛋白-1c (SREBP-1c)、脂肪酸合成蛋白(FAS)、脂肪分化相关蛋白(ADFP)的表达.各组TG含量测定行2×5的析因方差分析,两个样本均数比较用t检验,多个样本均数的比较用单因素多样本方差分析,多个样本间的两两比较用q检验.结果 实验组L02和HepG2细胞内油红O染色可见橘红色脂滴增多并出现融合现象,TG含量随着间歇性低氧天数的增加逐渐增多(Fl02=61.83,FHepG2=104.19,P值均＜0.01),氧浓度与时间有交互效应,差异有统计学意义(FL02=39.60,FHepG2=76.39,P值均＜0.01).ROS在对照组荧光显微镜下可见有基础表达,与对照组比较,间歇性低氧处理第2天,肝细胞内ROS荧光指数明显增加,差异有统计学意义(0.703±0.129与3.31±0.198,t02=22.0637;0.617±0.156与2.33±0.42,tHepG2=7.2003,P值均＜0.05),随着间歇性低氧处理时间延长,L02和HepG2细胞内ROS的含量增多(FL02=1021.84,FepG2=49.89,P值均＜0.01).Wesem blot结果显示,实验组HIF-1 α、SREBP-1c、FAS和ADFP蛋白相对表达量较对照组增加,并且随时间的延长逐渐增多,差异有统计学意义(L02细胞:FHIF-1α=371.19,FSREBP-1c=204.49,FFAS=38.2,FADFP=154.31,P值均＜0.05;HepG2细胞:FHIF-1α=150.84,FSREBP-1c=107.35,FFAS=279.71,FADFP=352.06,P值均＜0.01),HIF-2α蛋白相对表达量在实验组第1d较对照组增多(tL02=20.76,tHepG2=3.82,P值均＜0.05),此后逐渐降低至第4d低于对照组(tL02=3.18,tHepG2=2.54,P值均＜0.05).结论 间歇性低氧诱导产生的ROS可能调节低氧诱导因子和脂肪分化相关蛋白的表达,通过HIF-1 α-SREBP-1 c-FAS和ADFP途径导致肝细胞脂代谢异常.

abstractsObjective To determine the effect of intermittent hypoxia on lipid metabolism in liver cells and to explore the possible molecular pathways involved in this process.Methods An intermittent hypoxia cell model system was established by incubating the human hepatic cell lines L02 and HepG2 in an atmosphere of 2％ O2,5％ CO2 and 93％ N2 for 8 hours per day over a period of 1,2,3,4 or 5 days.Cells cultured in normoxia conditions (21％ O2) served as controls.Changes in intracellular lipid droplets and triglyceride (TG) levels were assessed by biochemical assays and oil red staining.Changes in intracellular reactive oxygen species (ROS) were assessed by inverted flurescence microscopy and flow cytometry.Changes in expression of hypoxia-inducible factor (HIF)-1α and HIF-2α proteins,and the downstream ADFP,SREBP-1c and FAS proteins,were detected by western blotting.Results For both L02 and HepG2 cell lines,the cells grown under hypoxic conditions showed significantly higher lipid droplet accumulation and TG content than the cells grown under normoxic conditions (FL02 =61.83,FHepG2 =104.19,P ＜ 0.01).Both oxygen concentration and time appeared to be correlated with these lipid-related changes (FL02 =39.60,FHepG2 =76.39,P ＜ 0.01).The ROS fluorescence index was significantly increased after 2 days of intermittent hypoxia (L02:0.703 ± 0.129 vs.3.310 ± 0.198,t =22.0637 and HepG2:0.617 ± 0.156 vs.2.33 ± 0.42,t =7.2003,P ＜ 0.05); in addition,increasing trends were observed in the ROS content and intensity of green fluorescence in conjunction with increased time of exposure to intermittent hypoxia (FL02 =1021.84,FHepG2 =49.89,P ＜ 0.01).Compared with their respective control groups,the L02 and HepG2 cells both showed significantly upregulated espression of HIF-1α ADFP,SREBP-1c and FAS （L02:FHIF-1α =371.19,FsREBP-1c =204.49,FFAS =38.20,FADFP =154.31,P ＜ 0.05 and HepG2:FHF-1α =150.84,FSRERBP-1c =107.35,FFAs =279.71,FADFP =352.06,P ＜ 0.01).In contrast,the HIF-2α level was markedly decreased in the cells after 4 and 5 days of exposure to intermittent hypoxia (FL02 =125.29,FHcpG2 =10.68,P ＜ 0.05).Conclusion Under intermittent hypoxic conditions,ROS may regulate the expression of hypoxia-inducible factors and the adipose differentiation-related protein,as well as influence fatty acid metabolism via a HIF-1 α-SREBP-1 c-FAS signal and upregulation of the ADFP protein,in liver cells.