【摘要】 探讨杂色曲霉素(sterigmatocystin,ST)对体外培养的永生化胃黏膜上皮细胞GES-1细胞周期的影响及细胞周期蛋白D1、真核起始因子eIF4E和其结合蛋白4E-BP1在ST诱导细胞周期变化中的可能作用。采用细胞培养、噻唑蓝(MTT)比色法、流式细胞定量检测(FCM)、吉姆萨(Giemsa)染色、蛋白质免疫印迹(Western印迹)以及反转录聚合酶联反应(RT-PCR)等方法,研究不同浓度(100、500、1000、2000μg/L)ST处理24h后,GES-1细胞的增殖、细胞周期分布及eIF4E、4E-BP1及细胞周期蛋白D1表达的变化情况。MTT法检测结果显示,ST可明显抑制GES-1细胞的增殖,ST100、500、1000、2000μg/L处理组的增殖抑制率分别为12.76%、16.35%、21.65%和32.06%。FCM检测结果显示,给予500、1000和2000μg/LST处理24h可剂量依赖性提高G2/M期细胞比例。吉姆萨染色结果表明,在0～2000μg/L的剂量范围内随ST剂量增高,细胞分裂指数逐渐降低,提示ST可诱导胃黏膜上皮细胞周期发生G2期阻滞。Western印迹分析结果显示,在0～2000μg/L浓度范围内,ST可剂量依赖性地上调eIF4E和4E-BP1在蛋白质水平上的表达(eIF4E:r=0.844,P<0.01;4E-BP1:r=0.930,P<0.01),而降低eIF4E和4E-BP1的磷酸化水平(peIF4E:r=-0.663,P<0.01;p4E-BP1:r=-0.656,P<0.01);同时使细胞周期蛋白D1的表达下降(r=-0.559,P<0.05)。RT-PCR检测在mRNA水平进一步证实了ST对eIF4E和4E-BP1表达的影响。研究结果表明,ST可抑制体外培养永生化胃黏膜上皮细胞增殖,诱导细胞发生G2期阻滞,4E-BP1表达升高,而eIF4E和4E-BP1的磷酸化水平降低及细胞周期蛋白D1表达下降可能是ST抑制胃黏膜上皮细胞增殖,诱导周期分布变化的重要机制。更多还原

【Abstract】 To explore the effects of sterigmatocystin (ST) on the cell cycle distribution and the expression of cyclinD1, eIF4E and 4E-BP1 of human gastric cell line (GES-1) in vitro. GES-1 cells were treated with ST at different concentrations (100, 500, 1 000 and 2 000 μg/L) for 24 h. The effects of ST on the cell proliferation of GES-1 cells were determined with MTT, flow cytometric (FCM) DNA analysis and Giemsa staining, while that on the expression of proliferation related gene——eIF4E, 4E-BP1 and cyclinD1 at protein level and mRNA level were studied with Western blot and reverse transcription polymerase chain reaction (RT-PCR) respectively. MTT results showed that ST treatment could significantly inhibit the proliferation of the GES-1 cells. The inhibition rate of ST 100, 500, 1 000 and 2 000 μg/L group was 12.76%, 16.35%, 21.65% and 32.06% respectively. FCM cell cycle analysis revealed that the G2/M fraction was significantly increased in 500, 1 000 and 2 000 μg/L ST treatment group 24 h after treatment. The results of Giemsa showed that within the concentration range from 0 to 2000 μg/L, ST could decrease the mitosis index (MI) of GES-1 dose-dependently. The FCM and Giemsa results suggest that ST could induce cell cycle G2 arrest on human gastric cells (GES-1) in vitro. Western blot analysis showed that ST could significantly increase the expression of eIF4E and 4E-BP1 within the concentration range from 0 to 2 000 μg/L, and there is a significant dose-effect correlation between ST concentration and the intensity of eIF4E and 4E-BP1 expression at protein level (eIF4E: r=0.844, P<0.01; 4E-BP1: r=0.930, P<0.01), while the phosphorylation of eIF4E and 4E-BP1 were significantly decreased by ST in a dose-dependent way (peIF4E: r=-0.663, P<0.01; p4E-BP1: r= -0.656, P<0.01). At the same time, the expression of cyclinD1 was down-regulated (r=-0.559, P<0.05). The results of RT-PCR confirmed that ST could increase the expression of eIF4E and 4E-BP1 at mRNA level of GES-1 cells in vitro. Thus, the results in this study suggested that ST could inhibit the proliferation and induce G2 arrest of human gastric GES-1 cells in vitro. The increase of eIF4E and 4E-BP1 and decrease of peIF4E, p4E-BP1 and cyclinD1 may be the putative mechanism.更多还原