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Ki-67启动子内Sp1位点与Hela细胞核蛋白结合活性
  • ISSN号:1001-9030
  • 期刊名称:《中华实验外科杂志》
  • 时间:0
  • 分类:R681[医药卫生—骨科学;医药卫生—临床医学;医药卫生—外科学]
  • 作者机构:[1]徐州医学院附属医院神经外科,江苏221002, [2]徐州医学院肿瘤生物治疗实验室
  • 相关基金:国家自然科学基金资助项目(30972976)
中文摘要:

目的 观察Ki-67启动子内能与细胞核蛋白结合的转录因子Sp1结合位点,探讨肿瘤细胞Ki-67基因高表达机制.方法 提取Hela细胞核蛋白.针对Ki-67启动子内4个潜在Sp1位点区域A1(-198~-172)、A2(-170~-144)、A3(-63~-37)、A4(-14~+12),以及没有Sp1结合位点的阴性对照区域C(-117~-91),设计相应的Sp1一致性寡核苷酸探针和Sp1突变性寡核苷酸探针,凝胶阻滞电泳实验(EMSA)验证Sp1探针与Hela细胞核蛋白的结合活性.结果 EMSA显示A2、A3、A4一致性探针均可以和Hela细胞核蛋白结合,这种结合可以被相应的100倍浓度非标记的一致性探针竞争抑制,不能被100倍浓度的非标记的突变性探针所抑制.出现结合带的反应中加入Sp1抗体后观察到明显的Supershift现象.A1和C相应探针不能和Hela细胞核蛋白发生结合反应.结论 Ki-67启动子-170~-144、-63~-37和-14~+12区域上存在能与肿瘤Hela细胞核蛋白结合的Sp1结合位点.

英文摘要:

Objective To investigate the transcription factors Spl binding sites of Ki-67 promoter that functions in the Ki-67 gene transcriptional regulation. Methods Electrophoretic mobility shift assay ( EMSA) was applied to verify the binding activity between putative Spl binding sites within Ki-67 promoter and nuclear protein of Hela cells. The nuclear extracts of HeLa cells were isolated and preincubated with and without an excess of unlabeled oligonucleotide competitors Spl probes in the presence or absence of antibody. Each reaction mixture was gel and electrophoresed. Results As indicated by EMSA, the consensus oligonucleotides A2 ( -170 to -144), A3 ( -63 to -37) , A4 ( - 14 to + 12) that contained the SP1 binding sites could bind to the nuclear extract of Hela cells. The binding could be competed by the 100 molar excess unlabeled consensus oligonucleotides competitor, but it could not be competed by the 100 molar excess unlabeled mute oligonucleotides competitor. The supershift bind could be observed when Spl antibody was added to the binding reaction. The consensus oligonucleotides Al and control oligonucleotides C could not bind to the nuclear extract of Hela cells. Conclusion These regions including - 170 to - 144, -63 to -37 and - 14 to +12 contained the Spl binding sites in the Ki-67 core promoter that could bind to the nuclear extracts of Hela cells specifically.

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期刊信息
  • 《中华实验外科杂志》
  • 北大核心期刊(2011版)
  • 主管单位:中国科学技术协会
  • 主办单位:中华医学会
  • 主编:
  • 地址:武汉市武昌区东湖路165号
  • 邮编:430064
  • 邮箱:cjes@cma.org.cn
  • 电话:027-87893475
  • 国际标准刊号:ISSN:1001-9030
  • 国内统一刊号:ISSN:42-1213/R
  • 邮发代号:38-85
  • 获奖情况:
  • 国内外数据库收录:
  • 美国化学文摘(网络版),日本日本科学技术振兴机构数据库,中国中国科技核心期刊,中国北大核心期刊(2004版),中国北大核心期刊(2008版),中国北大核心期刊(2011版),中国北大核心期刊(2014版),中国北大核心期刊(2000版)
  • 被引量:34996