CyclinB1的启动子在HeLa细胞cyclinB1蛋白调控中的作用.docVIP

Cyclin B1的启动子在Hela细胞cyclin B1蛋白调控中的作用 王昱1,2，卢仲义2，许峰2，朱宇迪3，朱宇熹4,5* 1 第三军医大学西南医院儿科 400038，2 重庆医科大学附属儿童医院PICU 400014，3 重庆医科大学儿童医院药剂科 400014，4 重庆医科大学附属第一医院肿瘤科 400016，5 Department of Radiation Oncology Image-applied Therapy Kyoto University Graduate School of Medicine，Japan [摘要] 　目的: 通过检测细胞周期素B1（cyclin B1）的启动子在Hela细胞的不同细胞周期的活性变化，探讨其对cyclin B1蛋白的调控作用。方法: 采用PCR法获得Hela细胞cyclin B1的启动子，通过基因重组插入pGL3 promoter vector从而获得质粒pGL3/cyclin B1 promoter。采用羟基脲（hydroxyurea HU）对Hela细胞进行细胞周期G1,S,G2/M期的同步化，通过流式细胞术法确认。用短暂转染的方法，将重组的质粒转染至Hela细胞。通过双萤光素酶活性检测分析cyclin B1的启动子在Hela细胞周期G1,S,G2/M期的活性的变化。结果: 双萤光素酶活性检测cyclin B1的启动子活性在G1期最高，S期最低，G2期中等。结论: 在Hela细胞中cyclin B1的启动子活性和cyclin B1蛋白的表达并不同步。 [关键词] 　细胞周期素B1; 　启动子，　Hela 细胞，　萤光素酶; The regulation of promoter of cyclin B1 in cyclin B1 protein Wang Yu1,2, Lu Zhongyi2, Xu Feng2，Zhu Yudi3， Zhu Yuxi4,5 1 Pediatric department，southwest hospital of the third military medical university，2 Department of PICU; children’s hospital affiliated to Chongqing medical university，3 Pharmacology department，children’s hospital affiliated to Chongqing medical university, 4 Department of Oncology，the first affiliated hospital of Chongqing medical university，5 Department of Radiation Oncology Image-applied Therapy Kyoto University Graduate School of Medicine，Japan Abstract: 　Objective 　To investigate the activity of cyclin B1 promoter in different cell cycle of Hela cell to explore its regulation on cyclin B1 protein. Methods 　The cyclin B1 promoter of Hela cell was obtained by PCR, and it was inserted into the plasmid of pGL3 promoter vector by recombination to construct the plasmid of pGL3/cyclin B1 promoter. Hela cells were synchronized in G1, S, G2/M phase by HU, which was proved by flow cytometry. The plasmid of pGL3/cyclin B1 promoter was transient transfected into Hela cells. The dual luciferase assay was performed in G1, S, G2/M phase. Results　The activity of cyclin B1 promoter was highest in G1 phase, decreased in S phase, and was medium in G2/M phase. Conclusion