摘要
为探讨Daxx对氧化型低密度脂蛋白(oxidized low-density lipoprotein,Ox-LDL)诱导巨噬细胞胆固醇蓄积和凋亡的介导作用及其可能的分子机制,用高效液相色谱法检测细胞内胆固醇含量,油红O染色观察细胞内脂滴的形成情况,流式细胞术和吖啶橙/溴化乙锭(AO/EB)染色法研究Ox-LDL对细胞凋亡的影响,Realtime RT-PCR检测细胞内Daxx mRNA的表达水平,Western blot检测caveolin-1蛋白的表达,用特异性siRNA沉默Daxx在RAW264.7细胞中的表达.Ox-LDL上调Daxx mRNA和caveolin-1的表达、增加细胞内胆固醇含量、促使RAW264.7细胞凋亡,用特异性siRNA干扰Daxx在RAW264.7细胞中的表达能降低caveolin-1的表达、减少细胞内胆固醇含量、以及抑制细胞凋亡.上述结果表明,Daxx对Ox-LDL诱导RAW264.7巨噬细胞胆固醇蓄积和凋亡具有介导作用,这一作用可能与Daxx上调caveolin-1的表达有关。
To explore whether Daxx mediates oxidized low-density lipoprotein（Ox-LDL）-induced cholesterol accumulation and apopotosis in macrophage and the underlying molecular mechanisms,intracellular lipid droplets and lipid levels were assayed by oil red O staining and high performance liquid chromatography（HPLC）,respectively,the apoptotic effect of RAW264.7 cells induced by Ox-LDL was analyzed by flow cytometric analysis and acridine orange/ethidium bromide（AO/EB） staining,the mRNA expressions of Daxx was quantified by Real time RT-PCR,the protein expression of caveolin-1 was detected by Western-blotting,Daxx-specific small interfering RNA（Daxx siRNA） was transfected to RAW264.7 cell by lipofectamin.Ox-LDL up-regulated the expression of Daxx mRNA,increased the accumulation of intercellular cholesterol in RAW264.7 macrophages,and induced the apoptosis of RAW264.7 macrophages.However,Ox-LDL-induced intercellular cholesterol accumulation and apoptosis in RAW264.7 cells was prevented by Daxx siRNA.Ox-LDL also induced caveolin-1 expression and this effect is significantly suppressed by Daxx siRNA.It can be concluded that Daxx mediates Ox-LDL-induced cholesterol accumulation and apoptosis in macrophages by up-regulating caveolin-1 expression.These findings provide an important demonstration that Daxx might be associated with the development of atherosclerosis.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2010年第8期881-890,共10页
Progress In Biochemistry and Biophysics
基金
supported by grants from The National Natural Science Foundation of China(30572191,30770868,30971267,30971170)~~
