CAJ | 학술논문

目的探讨孤儿核受体Nur77对巨噬细胞脂质沉积的影响及其机制.方法建市稳定表达绿色荧光蛋白(GFP)和GFP-Nur77质粒cDNA的小鼠巨噬细胞RAW264.7单克隆细胞系,40 μg/ml氧化型低密度脂蛋白(ox-LDL)刺激24 h后,用油红O染色定性观察细胞内脂质沉积,液相色谱-质谱联用法定量检测细胞内胆固醇酯,用荧光实时定量PCR(RT-PCR)检测CD36和腺苷三磷酸结合盒转运体A1(ABCA1)mRNA水平的变化,用流式细胞仪检测CD36蛋白表达,Western blot方法检测ABCA1蛋白表达.结果 Nur77能够显著减少ox-LDL诱导引起的巨噬细胞内脂质沉积.ox-LDL诱导前,GFP-RAW264.7组和GFP-Nur77-RAW264.7组细胞内总胆固醇含量分别为(38.66±0.13)μg/mg蛋白和(36.25±0.48)μg/mg蛋白,细胞内胆固醇酯含量为(18.85±0.03)μg/mg蛋白和(17.79±0.86)μg/mg蛋白.ox-LDL诱导24h后,GFP-RAW264.7组和GFP-Nur77-RAW264.7组细胞内总胆固醇含量分别为(68.98±1.68)μg/mg蛋白和(50.94±1.63)μg/mg蛋白,细胞内胆同醇酯含量为(35.92±2.28)μg/mg蛋白和(24.81±2.92)μg/mg蛋白.同时伴随CD36的mRNA及蛋白表达下降和ABCAI的mRNA及蛋白表达上调.结论孤儿核受体Nur77通过减少脂质摄取和增加脂质排出而减轻巨噬细胞内脂质沉积,这可能是其抗动脉粥样硬化的重要机制之一.
목적 탐토고인핵수체Nur77대거서세포지질침적적영향급기궤제.방법 건시은정표체록색형광단백(GFP)화GFP-Nur77질립cDNA적소서거서세포RAW264.7단극륭세포계,40 μg/ml양화형저밀도지단백(ox-LDL)자격24 h후,용유홍O염색정성관찰세포내지질침적,액상색보-질보련용법정량검측세포내담고순지,용형광실시정량PCR(RT-PCR)검측CD36화선감삼린산결합합전운체A1(ABCA1)mRNA수평적변화,용류식세포의검측CD36단백표체,Western blot방법검측ABCA1단백표체.결과 Nur77능구현저감소ox-LDL유도인기적거서세포내지질침적.ox-LDL유도전,GFP-RAW264.7조화GFP-Nur77-RAW264.7조세포내총담고순함량분별위(38.66±0.13)μg/mg단백화(36.25±0.48)μg/mg단백,세포내담고순지함량위(18.85±0.03)μg/mg단백화(17.79±0.86)μg/mg단백.ox-LDL유도24h후,GFP-RAW264.7조화GFP-Nur77-RAW264.7조세포내총담고순함량분별위(68.98±1.68)μg/mg단백화(50.94±1.63)μg/mg단백,세포내담동순지함량위(35.92±2.28)μg/mg단백화(24.81±2.92)μg/mg단백.동시반수CD36적mRNA급단백표체하강화ABCAI적mRNA급단백표체상조.결론 고인핵수체Nur77통과감소지질섭취화증가지질배출이감경거서세포내지질침적,저가능시기항동맥죽양경화적중요궤제지일.
Objective To observe the effect of Nur77 on lipid loading in macrophages exposed to 40 μg/ml oxidized low density iipoprotein (ox-LDL).Methods Stable RAW264.7 strain expressing green fluorescent protein (GFP) or GFP-Nur77 was established by G418 screening after transfection with corresponding plasmids and identified by Western blot.After 24 h stimulation with ox-LDL.intracellular lipid loading of each strain was observed by Oil Red O dyeing.and the intracellular cholesterol level was measured by liquid chromatographic-mass spectrometry (LC-MS).The transcriptional changes of CD36 and ABCA1 were monitored by Real Time Quantitative-PCR,while the expressions of these two proteins were assayed by flow cytometry and Western blot,respectively.Results After 24 h stimulation with ox-LDL,intracellular total cholesterol and esterified cholesterol concentration in GFP-Nur77-RAW264.7 were significantly dropped by 26.15% and 30.93% restxectively (P＜0.05 vs.GFP-RAW264.7).The transcription and expression of ABCA1 in GFP-Nur77-RAW264.7 were significantly increased while the transcription and expression of CD36 were significantly reduced (all P＜0.05 vs.GFP-RAW264.7).Conclusion Orphan nuclear receptor Nur77 reduced ox-LDL induced intracellular lipid loading in macrophages by inhibiting lipi d influx and enhancing lipid efflux.