转录因子环磷酸腺苷反应元件结合蛋白在紫杉醇诱导HeLa细胞周期阻滞中的作用及其机制  

Effects of transcription factor cAMP response element binding protein on taxol-induced HeLa cell-cycle arrest

作　　者：黄帅帅[1] 王雪[1] 庄海慧 王宇多 周细武[1] 王萍[1] 

机构地区：[1]宁波大学医学院解剖学与组织学胚胎学系,转化医学重点实验室,浙江宁波315211

出　　处：《解剖学报》2014年第4期485-492,共8页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(81372209,81071653);浙江省自然科学基金资助项目(LY13H160038)

摘　　要：目的探讨转录因子环磷酸腺苷反应元件结合蛋白(CREB)在紫杉醇诱导宫颈腺癌HeLa细胞周期阻滞中的作用及其分子机制。方法 MTT法确定紫杉醇的最佳浓度和处理时间;PCR技术构建pCI neo/CREB(PN)重组质粒及pCI neo/CREB-M(PM)定点突变质粒;流式细胞术检测细胞周期,Western blotting检测磷酸化CREB(pCREB)、CREB、cyclins以及CDKs蛋白表达。结果紫杉醇抑制HeLa细胞增殖的有效条件为0.1μmol/L处理24 h。0.1μmol/L紫杉醇诱导G2/M细胞数量增加,并呈时间依赖性;cyclin A表达量下调,cyclin B1、D1和pCREB表达量上调。此外,紫杉醇的处理对cyclin E、CDK1、CDK2、CDK4和CREB表达量并无显著性改变。然而,PM联合紫杉醇处理后显著性地反转了紫杉醇单独处理引起的cyclin A下调、cyclin B1和cyclin D1的上调,并且细胞周期G2/M期阻滞显著性减少。结论转录因子CREB介导的靶向细胞周期蛋白表达在紫杉醇诱导的细胞周期阻滞过程中扮演重要角色。Objective To explore the effects of cAMP response element binding protein （ CREB） on taxol-induced cell cycle arrest in HeLa cells .Methods MTT assay was used to determine the optimal concentration and treatment time . PCR method was used to construct the recombinant plasmid pCI neo /CREB（ PN） and site-directed mutagenesis recombinant plasmid pCI neo/CREB-M（PM）.Cell cycle was assayed by flow cytometry .Expressions of pCREB, CREB, cyclins and CDKs were assayed by Western blotting .Results The effective conditions of taxol treatment on HeLa cells were 0.1μmol/L for 24 hours.After cells were treated with 0.1μmol/L taxol, G2/M phase was arrested in a time-dependent manner , accomplished with the decrease of cyclin A , a significant increase of cyclin B1, D1 and phosphorylated CREB （pCREB） protein expression, whereas, no marked changes were observed in cyclin E , CDK1, CDK2, CDK4 and CREB expressions. However, combinantion of PM and taxol treatment significantly reduced taxol-induced G2/M phase arrest, and reversed the effect of taxol-decreased cyclin A, increased cyclin B1 and D1 expression.Conclusion Tanscription factor CREB-mediated specific cyclins play a pivotal role in taxol-induced G 2/M arrest in HeLa cells .

关 键 词：宫颈癌 环磷酸腺苷反应元件结合蛋白 细胞周期 流式细胞术 免疫印迹法 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]