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.
目的 探討間歇性低氧對肝細胞脂質代謝的影響及其機製.方法 利用三氣培養箱(O2、CO2、N2體積分數分彆為2％、5％、93％)8 h/d間歇性低氧處理L02和HepG2細胞1、2、3、4、5d,建立間歇性低氧細胞模型,油紅O染色及甘油三酯(TG)測定檢測肝細胞內的脂滴含量,流式細胞儀及熒光顯微鏡觀察間歇性低氧對肝細胞內活性氧(ROS)的影響,Western blot檢測ROS對低氧誘導因子(HIF)-1α和HIF-2 α蛋白錶達的調節,Western blot檢測HIF-1α下遊與脂質代謝相關的固醇調節元件結閤蛋白-1c (SREBP-1c)、脂肪痠閤成蛋白(FAS)、脂肪分化相關蛋白(ADFP)的錶達.各組TG含量測定行2×5的析因方差分析,兩箇樣本均數比較用t檢驗,多箇樣本均數的比較用單因素多樣本方差分析,多箇樣本間的兩兩比較用q檢驗.結果 實驗組L02和HepG2細胞內油紅O染色可見橘紅色脂滴增多併齣現融閤現象,TG含量隨著間歇性低氧天數的增加逐漸增多(Fl02=61.83,FHepG2=104.19,P值均＜0.01),氧濃度與時間有交互效應,差異有統計學意義(FL02=39.60,FHepG2=76.39,P值均＜0.01).ROS在對照組熒光顯微鏡下可見有基礎錶達,與對照組比較,間歇性低氧處理第2天,肝細胞內ROS熒光指數明顯增加,差異有統計學意義(0.703±0.129與3.31±0.198,t02=22.0637;0.617±0.156與2.33±0.42,tHepG2=7.2003,P值均＜0.05),隨著間歇性低氧處理時間延長,L02和HepG2細胞內ROS的含量增多(FL02=1021.84,FepG2=49.89,P值均＜0.01).Wesem blot結果顯示,實驗組HIF-1 α、SREBP-1c、FAS和ADFP蛋白相對錶達量較對照組增加,併且隨時間的延長逐漸增多,差異有統計學意義(L02細胞:FHIF-1α=371.19,FSREBP-1c=204.49,FFAS=38.2,FADFP=154.31,P值均＜0.05;HepG2細胞:FHIF-1α=150.84,FSREBP-1c=107.35,FFAS=279.71,FADFP=352.06,P值均＜0.01),HIF-2α蛋白相對錶達量在實驗組第1d較對照組增多(tL02=20.76,tHepG2=3.82,P值均＜0.05),此後逐漸降低至第4d低于對照組(tL02=3.18,tHepG2=2.54,P值均＜0.05).結論 間歇性低氧誘導產生的ROS可能調節低氧誘導因子和脂肪分化相關蛋白的錶達,通過HIF-1 α-SREBP-1 c-FAS和ADFP途徑導緻肝細胞脂代謝異常.
목적 탐토간헐성저양대간세포지질대사적영향급기궤제.방법 이용삼기배양상(O2、CO2、N2체적분수분별위2％、5％、93％)8 h/d간헐성저양처리L02화HepG2세포1、2、3、4、5d,건립간헐성저양세포모형,유홍O염색급감유삼지(TG)측정검측간세포내적지적함량,류식세포의급형광현미경관찰간헐성저양대간세포내활성양(ROS)적영향,Western blot검측ROS대저양유도인자(HIF)-1α화HIF-2 α단백표체적조절,Western blot검측HIF-1α하유여지질대사상관적고순조절원건결합단백-1c (SREBP-1c)、지방산합성단백(FAS)、지방분화상관단백(ADFP)적표체.각조TG함량측정행2×5적석인방차분석,량개양본균수비교용t검험,다개양본균수적비교용단인소다양본방차분석,다개양본간적량량비교용q검험.결과 실험조L02화HepG2세포내유홍O염색가견귤홍색지적증다병출현융합현상,TG함량수착간헐성저양천수적증가축점증다(Fl02=61.83,FHepG2=104.19,P치균＜0.01),양농도여시간유교호효응,차이유통계학의의(FL02=39.60,FHepG2=76.39,P치균＜0.01).ROS재대조조형광현미경하가견유기출표체,여대조조비교,간헐성저양처리제2천,간세포내ROS형광지수명현증가,차이유통계학의의(0.703±0.129여3.31±0.198,t02=22.0637;0.617±0.156여2.33±0.42,tHepG2=7.2003,P치균＜0.05),수착간헐성저양처리시간연장,L02화HepG2세포내ROS적함량증다(FL02=1021.84,FepG2=49.89,P치균＜0.01).Wesem blot결과현시,실험조HIF-1 α、SREBP-1c、FAS화ADFP단백상대표체량교대조조증가,병차수시간적연장축점증다,차이유통계학의의(L02세포:FHIF-1α=371.19,FSREBP-1c=204.49,FFAS=38.2,FADFP=154.31,P치균＜0.05;HepG2세포:FHIF-1α=150.84,FSREBP-1c=107.35,FFAS=279.71,FADFP=352.06,P치균＜0.01),HIF-2α단백상대표체량재실험조제1d교대조조증다(tL02=20.76,tHepG2=3.82,P치균＜0.05),차후축점강저지제4d저우대조조(tL02=3.18,tHepG2=2.54,P치균＜0.05).결론 간헐성저양유도산생적ROS가능조절저양유도인자화지방분화상관단백적표체,통과HIF-1 α-SREBP-1 c-FAS화ADFP도경도치간세포지대사이상.
Objective 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.
