低剂量三氧化二砷对HepG2细胞p53基因转录水平的下调作用
三氧化二砷,,三氧化二砷;p53基因启动子;反式激活;下调,1材料和方法,2结果,3讨论,参考文献:
(新疆医科大学公共卫生学院, 新疆乌鲁木齐830054; 北京地坛医院, 北京100011; 解放军第302医院传染病研究所基因治疗研究中心, 北京100039)摘要: 目的:探讨低剂量三氧化二砷(As2O3)对p53启动子转录活性的调节作用。方法:确定p53启动子区域,聚合酶链反应(PCR)扩增p53p,克隆至真核报告载体pCAT3Basic中,构建pCAT3p53p报告载体;以该质粒转染肝癌细胞系HepG2细胞系,用酶联免疫吸附法(ELISA)检测氯霉素乙酰转移酶(CAT)的表达活性;并以不同浓度三氧化二砷刺激HepG2细胞系,用ELISA法检测CAT的表达活性。结果: 成功克隆出614 bp的新的p53启动子, pCAT3p53p和不同浓度的三氧化二砷(0.25 μmoL/L)瞬时转染的HepG2细胞的CAT表达活性是pCAT3Basic空载体的11.2倍,表明pCAT3p53p具有强的启动子活性。选用0.25、0.5、2.5、5.0 μmol/L浓度的三氧化二砷刺激pCAT3 p53p转染的HepG2细胞,其CAT吸光度值分别为:0.076、0.186、0.149、0.119, CAT活性随着三氧化二砷浓度的倍增呈衰减趋势。结论: p53启动子有反式激活下游基因表达的活性,低剂量三氧化二砷对p53基因转录水平具有下调作用。
关键词: 三氧化二砷;p53基因启动子;反式激活;下调
Downregulation of transcriptional activation of p53 gene promoter by low dosage arsenic trioxide
WU Shunhua, ZHENG Yujian, CHENG Jun, et al
(Department of Labour and Environment Hygiene, College of Public Health, Xinjiang Medical University, Urumqi 830054, China)
Abstract: Objective: To investigate the transcriptional activation of p53 gene promoter by low dosage arsenic trioxide (As2O3). Methods: Polymerase chain reaction (PCR) technique was employed to amplify the coding sequence of p53 promoter from HepG2 genomic DNA, and the product was subcloned into pCAT3Basic by Kpn I and Xho I, named pCAT3p53p. pCAT3p53p was transfected into the hepatoblastoma cell line HepG2, then cotransfected with As2O3 by FuGENE 6 transfection reagents. The HepG2 cells transfected with pCAT3Basic was used as negative control. The activity of promoter region fused to the CAT gene in HepG2 cells transfected was detected by a ELISA kit after 48 hours, which reflected the transregulating function of As2O3 to p53p gene promoter. Results: The report vector pCAT3 p53p had been constructed and had been confirmed by restriction enzyme digestion and sequencing. The expression of CAT in HepG2 cells cotransfected with pCAT3 p53p and As2O3 was respectively 0.076 ......
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