摘要
目的探讨双环醇对细胞周期素(cyclin)B2启动子转录活性的调节作用。方法根据文献报道的结果确定细胞周期素B2的启动子DNA序列区域,以聚合酶链反应(PCR)扩增细胞周期素启动子(B2p),克隆至真核报告载体pCAT3Basic中,构建pCAT3cyclinB2p报告载体;以该质粒转染肝癌细胞系HepG2细胞,用酶联免疫吸附法(ELISA)检测氯霉素乙酰转移酶(CAT)的表达活性并与双环醇共刺激的HepG2细胞,用ELISA法检测CAT的表达活性。结果成功获得细胞周期素B2启动子的止确克隆。pCAT3cyclinB2p和双环醇(106Mol/L)瞬时转染的HepG2细胞的CAT表达活性是pCAT3Basic空载体的2.4倍,pCAT3cyclinB2p的0.35倍。结论细胞周期素B2启动子有顺式激活下游基因的活性,双环醇具有对细胞周期素B2基因有下调作用。
Objective To investigate the trans-regnlating effect of bicyclol on cyclin B2 gene promoter. Methods Polymerase chain reaction （PCR） technique was employed to amplify the sequence of cyclin B2 promoter by using HepG2 genomic DNA as template, and the amplified product was subcloned into pCAT3-Basic at Kpn I and Bgl 11 sites, and the resulted plasmid was designated pCAT3-cyclin B2p. pCAT3-cyclin B2p was transfected into the hepatoblastoma cell line HepG2 by FuGENE 6 transfection re agents, then stimulated with bicyclol. The HepG2 cells transfected with pCAT3-Basic was used as negative control. The activity of CAT inHepG2 cells transfected was detected by an enzyme-linked immunosorbent assay（ELISA） kit after 48 hours, which reflected the trans-regnlating function of bicyclol on eyclin B2p gene promoter. Results The report vector pCAT3-cyclin B2p has been constructed and had been confirmed by restriction enzyme digestion and sequencing. The expression of CAT in HepG2 cells co-transfected with pCAT3-cyclin B2p and bicyclol was 2.4 times as higher as that of pCAT3-Basic, and 0.35 as higher as that of pCAT3-cyclin B2p. Conclusion Bicyclol candown-regnlate cyclin B2 promoter.
出处
《胃肠病学和肝病学杂志》
CAS
2005年第4期346-348,共3页
Chinese Journal of Gastroenterology and Hepatology
基金
国家自然科学基金攻关项目(C03011402
C30070689)
军队九五科技攻关项目(98D063)
军队回国留学人员启动基金项目(98H038)
军队十五科技攻关青年基金项目(01Q138)
军队十五科技攻关项目(01MB135)
