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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。
We screened and analyzed the composition of GCN5 recruitment and differentiation related protein aggregates during the differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes. Immunofluorescence cytochemistry and real-time fluorescence quantitative PCR were used to identify the MSCs differentiated into cardiomyocyte-like cells by 5-azacytidine (5-azaC). The co-immunoprecipitation and MS / MS were used to identify the composition of GCN5- Screening from the perspective of cardiomyocyte differentiation to verify the differentiation-related protein factor. The cardiac myocyte-like genes GATA4, MEF2C and cardiomyocyte structure, functional proteins cTnt, MHC and Cx43 were induced by 5-azaC-induced cardiomyocyte-like cells. Screening and identification of cardiac myocyte differentiation-related GCN5-recruited proteins were classified as: DNA binding protein Sp1 / KLF; (2) transcriptional coactivators PGC-1α and Rb1; (3) transcription elongation complex components and signaling pathway protein Akt. Through screening, we found that MSCs can differentiate into cardiomyocyte-like cells during the process of cardiomyocyte-like differentiation, which lays the foundation for the further study of stem cell differentiation signal transduction pathway, specific biological target intervention and stem cell differentiation efficiency.