基础医学与临床 ›› 2010, Vol. 30 ›› Issue (7): 689-697.

• 研究论文 • 上一篇    下一篇

组蛋白乙酰化酶调控MSCs经5-azaC诱导后的细胞周期和增殖特性

周娜 朱静 田杰 张亚兰 邓兵 李娅莎   

  1. 重庆医科大学附属儿童医院 重庆医科大学附属儿童医院 重庆医科大学附属儿童医院
  • 收稿日期:2009-09-07 修回日期:2010-03-02 出版日期:2010-07-05 发布日期:2010-07-05
  • 通讯作者: 朱静

Histone acetyltransferase regulates cell cycle and cell proliferation of mesenchymal stem cells induced by 5-azacytidine

Na ZHOU, Jing ZHU, Jie TIAN, Ya-lan ZHANG, Bing DENG, Ya-sha LI   

  1. Children,s Hospital, Chongqing Medical University Pediatrics Institute; Chongqing University of Medical Science
  • Received:2009-09-07 Revised:2010-03-02 Online:2010-07-05 Published:2010-07-05
  • Contact: Jing ZHU,

摘要: 目的 阐述组蛋白乙酰化酶GCN5在间充质干细胞经5-氮杂胞苷诱导过程中的作用及其转录调控机制。方法 MTT法、流式细胞术检测细胞周期和细胞增殖;定量PCR检测P21基因表达;CHIP技术验证GCN5募集蛋白复合体与P21基因的相互作用及P21基因启动子区域的乙酰化水平;Co-IP技术分离、串联质谱技术鉴定GCN5募集蛋白复合体组成。结果 MSCs经5-azaC诱导3 d后G0/G1期细胞比例、P21基因表达量最高,此后逐渐降低,细胞增殖指数及G2/S期细胞比例与上述结果相反;筛选出GCN5募集蛋白复合体为:依赖ATP的染色质重塑复合体成分、转录起始复合体成分、转录因子和锌指结构蛋白;诱导组GCN5与P21基因启动子区域结合能力及P21基因启动子区域组蛋白H3乙酰化水平高于未诱导组。结论 GCN5募集蛋白复合体通过结合于P21基因启动子区域参与调控MSCs经5-azaC诱导体外分化过程中细胞周期G0/G1期和细胞增殖特性的调控。

关键词: 骨髓间充质干细胞, 组蛋白乙酰化酶GCN5, 生物学特性, 免疫共沉淀, 串联质谱

Abstract: Objective The purpose of the experiment is explaining the role of histone acetyltransferase GCN5 in the process of mesenchymal stem cells induced by 5-azacytidine and the regulation mechanism of GCN5 complex. Methods Cell proliferation was evaluated by MTT assay, cell cycle by flow cytometry and expression of P21 by real-time PCR. Then we used co-immunoprecipitation strategy to separate interaction proteins with GCN5 and tandem mass spectrometry to identify interacting proteins. CHIP strategy was used for analyzing binding ability between P21 promoter and GCN5 together with acetylated histones at the GCN5 site on the P21 gene. Results The proportion of G0/G1 phase MSCs and expression of P21 gene reached the highest at 3 days after 5-azaC-induced, then gradually decreasing. Cell proliferation index PI value and the G2/S phase cell ratio were the opposite to the above. GCN5 physically interacted with the following proteins which were categorized as:ATP-dependent chromatin remodeling complex、transcription initiation complex、transcription factor and zinc finger protein. Binding-abilities and levels of acetylation of 5-azaC treated group were higher than that of untreated group. Conclusion High concentration of 5-azaC increases G0/G1 phase arrest for MSCs. G0/G1 phase arrest decreased and cell proliferation increased as the concentration of 5-azaC decreasing. 5-azaC raised the combination of GCN5-protein-complexes and the P21 gene promoter in vitro differentiation of MSCs through regulating G0/G1 phase of cell cycle and cell proliferation.

Key words: Mesenchymal Stem Cells, histone acetyltransferase GCN5, Biological Characteristics, Co-IP, MS/MS