摘要
目的通过组蛋白乙酰化作用相关shRNA质粒转染5氮杂胞苷(5-azacytid ine,5-aza)诱导后不同时间段间充质干细胞心肌发育基因的检测,探讨乙酰化修饰在干细胞特化心肌样细胞转录调控中的作用。方法选择7条组蛋白乙酰化修饰关键因子Gcn5基因片段,用带有绿色荧光蛋白(green fluorescence prote in,GFP)基因的pGenesil-1作为载体,构建相应7个shRNA质粒。5-aza诱导大鼠间充质干细胞(m esenchym al stem cells,MSCs)24 h、2、4、7 d,分别用重组shRNA质粒脂质体共转染后提取RNA,逆转录PCR检测Gcn5基因和心肌早期发育基因GATA4的表达。结果经特异性限制性内切酶酶切分析和DNA序列测定,质粒构建与设计符合;各个时间段对照组Gcn5和GATA4均有表达,而实验组却无相应表达。结论通过实验提示封阻干细胞染色质组蛋白乙酰化可以达到抑制干细胞特化心肌细胞转录过程的作用,这为进一步研究组蛋白末端乙酰化修饰与干细胞分化调节机制奠定实验室基础。
Objective To construct 7 shRNA plasmids targeted to Gcn5 and to explore their role on histone acetylation in regulating MSCs differentiation into cardiomyocytes. Methods MSCs were isolated and passaged. Seven shRNA plasmids were constructed by pGenesil-1 （4.9 kb ） vector with GFP gene. MSCs were treated with 10μmol/L 5-azacytidine respectively for 24 h, 2, 4 and 7 d and transfected with the plasmids via lipofectamine^TM2000. Gcn5, as the marker of the acetylation, and GATA4, as the marker of cardiomyocytes, were detected in MSCs during differentiation with or without shRNA plasmids transfection by RT-PCR. Results MSCs were fibroblast-like but changed morphologically after induction. Seven Gcn.5 shRNA plasmids were constructed and identified by restriction endonucleases Psi Ⅰ/Sal Ⅰ and DNA sequencing. The expression of Gcn5 and GATA4 could be detected in MSCs without shRNA plasmids tranfection, but no expression after shRNA plasmids transfection. Conclusion Gcn5 plays a very important role in MSCs differentiation into cardiomyocytes, which lays the basis for further research of the importance of acetylation in the regulation of cell differentiation.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第6期535-538,共4页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目(30471837)~~
