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  

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

作　　者：谢超[1] 王方圆[2] 袁运生[1] 郜尽[3] 朱润芝 韩伟[3] 俞雁[1] 

机构地区：[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　　处：《上海交通大学学报（医学版）》2011年第5期551-555,560,共6页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家高技术研究发展计划("八六三"计划)(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。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. ConclusionProtein 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.

关 键 词：肝脏再生 转录因子 酵母双杂交系统 蛋白互作 

分 类 号：Q78[生物学—分子生物学]