›› 2011, Vol. 31 ›› Issue (5): 551-.doi: 10.3969/j.issn.1674-8115.2011.05.006

• 论著(基础研究) • 上一篇    下一篇

酵母双杂交研究小鼠肝脏再生TGF-β调控通路中相关转录因子的相互作用

谢 超1, 王方圆2, 袁运生1, 郜 尽3, 朱润芝4, 韩 伟3, 俞 雁1   

  1. 1.上海交通大学 农业与生物学院上海市兽医生物技术重点实验室, 上海 200240; 2.上海交通大学 医学院附属中国福利会国际和平妇幼保健院中心实验室, 上海 200030; 3.上海交通大学 药学院, 上海 200240; 4.中国药科大学江苏省肿瘤发生与干预重点实验室, 南京 210009
  • 出版日期:2011-05-28 发布日期:2011-05-27
  • 通讯作者: 俞 雁, 电子信箱: yanyu@sjtu.edu.cn。
  • 作者简介:谢 超(1982—), 男, 博士生;电子信箱: xiechao@sjtu.edu.cn。
  • 基金资助:

    国家高技术研究发展计划(“八六三”计划)(2007AA02Z149)

Transcription factor interactions of TGF-beta signaling pathway identified by yeast two-hybrid technique during mice liver regeneration

XIE Chao1, WANG Fang-yuan2, YUAN Yun-sheng1, GAO Jin3, ZHU Run-zhi4, HAN Wei3, YU Yan1   

  1. 1.Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, China;2.Center of Research Laboratory, the International Peace Maternity and Child Health Hospital of China Welfare Institute, Shanghai Jiaotong University School of Medicine, Shanghai 200030, China;3.School of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, China;4.Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing 210009, China
  • Online:2011-05-28 Published:2011-05-27
  • Supported by:

    National High-tech R&D Program of China, 2007AA02Z149

摘要:

目的 采用酵母双杂交系统研究肝脏再生调控途径转化生长因子β(TGF-β)信号通路中4种转录因子的相互作用。方法 经CCl4腹腔注射诱导建立小鼠肝损伤再生模型,提取模型小鼠肝脏组织总RNA作为模板,经RT-PCR获得cDNA。经肝脏再生基因芯片检测显示差异表达且与TGF-β信号通路相关的激活转录因子3(ATF3)、DNA结合抑制因子3(ID3)、DNA损伤诱导的转录因子3(DDIT3)和LIM蛋白家族成员FHL2(FHL2)作为候选基因,进行基因克隆和载体构建,PCR及DNA测序鉴定。采用酵母双杂交系统进行自激活检测实验和酵母双杂交实验,观察ATF3、FHL2、DDIT3和ID3之间的相互作用。结果 PCR及DNA测序鉴定表明,ATF3、FHL2、DDIT3、ID3基因克隆与载体构建成功。自激活检测显示ATF3、FHL2、DDIT3和ID3不存在自激活现象;酵母双杂交实验发现7对蛋白互作,分别为ID3/ATF3、FHL2/ATF3、ATF3/ATF3、FHL2/FHL2、ID3/FHL2、ID3/ID3和ID3/DDIT3。结论 ATF3、FHL2和ID3间的蛋白互作可能对TGF-β信号通路具有调控作用;蛋白互作网络可作为肝损伤再生机制研究的途径之一。

关键词: 肝脏再生, 转录因子, 酵母双杂交系统, 蛋白互作

Abstract:

Objective To investigate the interactions of four transcription factors of transforming growth factor-β (TGF-β) signaling pathways using yeast two-hybrid technique. Methods CCl4-induced liver injury mouse models were established, and the total RNA of liver tissues were extracted as the template for cDNA by RT-PCR. Activating transcription factor 3 (ATF3), inhibitor of DNA binding 3 (ID3), DNA-damage-inducible transcript 3 (DDIT3) and LIM domains 2 (FHL2), which were differentially expressed detected by liver regeneration gene chip and associated with TGF-β signaling pathway, were served as candidate genes. After gene clone, vector construction and PCR and DNA sequencing, yeast two-hybrid system was used to indentify the interactions among ATF3, ID3, DDIT3 and FHL2 with auto-activation test. Results PCR and DNA sequencing indicated that ATF3, FHL2, DDIT3 and ID3 were successfully cloned, and there was no auto-activation phenomenon after auto-activation test. Seven protein interactions (ID3/ATF3, FHL2/ATF3, ATF3/ATF3, FHL2/FHL2, ID3/FHL2, ID3/ID3 and ID3/DDIT3) were identified by yeast two-hybrid technique. Conclusion Protein interactions among ATF3, FHL2 and ID3 may be one of the possible ways to regulate the TGF-β signaling pathway, and protein-protein interactions may be a key gateway to liver regeneration.

Key words: liver regeneration, transcription factors, yeast two-hybrid system, interactions