人白血病细胞HL-60凋亡启动时相的亚细胞蛋白质组学研究

Subcellular Proteomic Analysis of the Startup Phase of Apoptosis of the HL-60 Cells Induced by All-trans Retinoic Acid

目的:建立全反式维甲酸(ATRA)诱导HL-60细胞凋亡启动模型,分析凋亡启动时细胞总蛋白和亚细胞组分蛋白的表达情况。方法:体外培养HL-60细胞,氮唑蓝(MTT)比色法检HL-60细胞测增殖活性,流式细胞术(FCM)检HL-60细胞测细胞凋亡和细胞周期分布;高速离心法分离对照组与凋亡启动时相的HL-60细胞核、膜和细胞器以及细胞质等组分蛋白;双向电泳(2-DE)HL-60细胞总蛋白和亚细胞组分蛋白,图像分析软件分析2-DE图谱,筛选差异表达蛋白,并进行液相色谱串联质谱(LC-MS/MS)鉴定。结果:ATRA明显抑制HL-60细胞增殖,并随浓度、时间的增加,抑制作用也越显著;以1μmol/L的ATRA诱导HL-60细胞48 h早期凋亡比例为13.21%,细胞形态也存在明显变化;筛选出22个差异表达蛋白点,其中5个蛋白在凋亡启动时表达上调,15个蛋白表达下调,2个蛋白只在凋亡启动时表达;它们的功能主要涉及与细胞凋亡相关的蛋白,与细胞分化、增殖相关的蛋白和其它蛋白。结论:1μmol/L的ATRA诱导HL-60细胞生长48 h,成功建立细胞凋亡启动模型;凋亡启动时相的HL-60细胞质、膜和细胞器以及细胞总蛋白的表达都存在差异,这些差异表达蛋白可能在细胞凋亡启动时起重要作用。

Objective：To establish the the HL-60 cell apoptosis startup model induced by all-trans retinoic acid（ ATRA）and investigate the different expression of total protein and subcellular fraction of HL-60 cells. Methods：HL 60 cells were cultured in vitro,MTT and flow cytometry were used to de-tect the HL60 cell proliferation and the cell cycle distribution. Ultracentrifugation was adopted to sepa-rate fraction protein such as nucleus and cytoplasm components,membrane and organelles of the con-trol group and HL-60 cells of apoptosis startup phase. Adopting image analysis software to analyze the two-dimensional electrophoresis（2-DE）for screening the differentially expressed protein between trea-ted and untreated HL-60 cells. The protein was identified by liquid chromatography tandem mass spec-trometry（ LC-MS/MS）. Results：ATRA obviously inhibited HL-60 cell proliferation,and with the in-crease of concentration and exposing time,the inhibiting effect was enhanced. Treated with 1 μmol/L ATRA for 48 h,13. 21% of HL-60 cells apoptosis occurred and the cell morphology changed. 22 dif-ferentially expressed protein spots were screened,with which 5 of them were high-regulation of expres-sion,15 of them were down-regulation of expression,2 of them were expressed only at startup phase of apoptosis. Their functions mainly involved the following metabolic pathways：cell apoptosis related pro-teins,proteins associated with cell differentiation,proliferation,and other proteins. Conclusions：HL-60 cells are induced by ATRA in 1 μmol/L concentration for 48 h,the apoptosis startup phase model is successfully established. There are differentially expressed proteins of subcellular components be-tween control and apoptosis initiation HL-60 cells,these proteins also played an important role in the startup of apoptosis.