杂色曲霉素诱导胃黏膜上皮细胞细胞周期G_2期阻滞  

Sterigmatocystin Induced G_2 Arrest of Gastric Epithelial Cells in Vitro

作　　者：邢欣[1] 邢凌霄[1] 李月红[1] 王娟[1] 姚志刚[1] 王俊灵[1] 刘亚玲[1] 张祥宏[1] 

机构地区：[1]河北医科大学病理研究室,石家庄市050017

出　　处：《细胞生物学杂志》2009年第1期89-95,共7页Chinese Journal of Cell Biology

基　　金：河北省自然科学基金(No.C2007000819);教育部科学技术研究重点项目(No.205021);河北省科技攻关研究重点项目(No.07276102D)资助~~

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。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 000μg/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 tag/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 000μgg/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-BPI： 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-BPI： 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

关 键 词：杂色曲霉素 EIF4E 4E—BP1 细胞周期蛋白D1 

分 类 号：R363[医药卫生—病理学]