摘要
筛选并分析间充质干细胞(mesenchymal stem cells,MSCs)诱导分化为心肌样细胞过程中GCN5招募促分化相关蛋白集合体的组成。免疫荧光细胞化学、实时荧光定量PCR鉴定MSCs经5-氮杂胞苷(5-azacytidine,5-azaC)诱导分化为心肌样细胞;免疫共沉淀技术分离、串联质谱鉴定GCN5募集蛋白集合体组成,并从心肌细胞分化角度筛选、验证分化相关蛋白因子。MSCs经5-azaC诱导分化的心肌样细胞表达心肌特异性基因GATA4、MEF2C和心肌细胞结构、功能蛋白cTnt、MHC和Cx43;筛选、验证出心肌细胞分化相关GCN5招募蛋白归类为:(1)DNA结合蛋白Sp1/KLF;(2)转录辅激活子PGC-1α和Rb1;(3)转录延伸复合体组成成分以及信号通路蛋白Akt。通过筛选获得MSCs经诱导向心肌样细胞分化过程中心肌分化相关蛋白因子,为进一步研究干细胞分化信号传导途径、特异性生物靶点干预以及提高干细胞分化效率打下了基础。
Screening and analysing the recruitment of protein complexes with GCN5 during the process of mesenchymal stem cells （MSCs） differentiating into cardiac myocytes have been designed to identify unknown proteins that were recruited by histone acetyltransferase GCN5. Immunofluorescence cytochemistry and real-time PCR analysis identify that MSCs can differentiate into cardiomyocyte-like cells after 5-azaC induction treatment. We then developed co-immunoprecipitation strategy to separate recruitment proteins with GCN5, and tandem mass spectrometry to identify recruitment proteins. Furthermore, we analyzed functions of GCN5 recruitment proteins from the degree of differentiation of cardiac. MSCs can express higher cardiac-specific genes GATA4 and MEF2C after 5-azaC induction treatment. GCN5 physically interacts with the following proteins which can be categorized as： （1） DNA-binding protein of Spl/KLF; （2） transcriptional co-activators of PGC-1α and Rbl; （3） components of transcription elongation complex, and finally signaling pathway component of protein Akt. This experiment screens GCN5 recruitment proteins for regulation. We will further study differentiating signal transduction pathway of stem cells, as well as the specific biological target for intervention to improve the ratio of differentiation of stem cells on the basis of exoeriments.
出处
《中国细胞生物学学报》
CAS
CSCD
2010年第1期61-68,共8页
Chinese Journal of Cell Biology
基金
国家自然科学基金资助项目(No.30973219)~~
