【摘要】 ISL1是第二生心区的分子标志,在心血管发育中发挥重要作用.在心脏发育过程中,Isl1的表达具有鲜明的时空特异性.本研究利用P19CL6畸胎瘤干细胞作为心肌分化模型,探讨了Isl1在心肌诱导分化过程中的时间特异性表达及经典Wnt信号通路对其的调控.研究发现,Isl1在心肌分化早期高表达,于诱导第4 d到达高峰,随后快速下调.其表达趋势与经典Wnt通路的激活模式具有时间上的同步性.通过加入Wnt3a蛋白及Li Cl激活经典Wnt通路,能够促进Isl1基因的表达,而Wnt通路抑制分子Frizzled-4/Fc和DKK1能够下调Isl1表达.β-catenin过表达及RNAi实验也获得相似的结果.染色质免疫共沉淀实验证实,Wnt通路效应分子LEF1,在细胞分化第4 d与其在Isl1基因启动子上游-2 300 bp处的结合增强,因而促进了Isl1基因的表达.本研究表明,经典Wnt信号能够通过LEF1/β-catenin与Isl1启动子特异结合,调控Isl1基因在心肌早期分化阶段的表达.更多还原

【Abstract】 Insulin gene enhancer protein 1( ISL1),a marker of the second heart field,displays a temporal-spatial expression profile and plays crucial roles during cardiac development. This study aimed to investigate the kinetics of Isl1 expression at different differentiation stages of P19CL6 cells into cardiomyocytes. The regulatory effect of canonical Wnt signaling on Isl1 expression was characterized.The level of Isl1 mRNA was elevated following DMSO induction,peaked at day 4,and with a attenuated sharp decline afterward. The expression of Isl1 fitted the activation pattern of canonical Wnt signaling triggered by Wnt3 a and Li Cl. The Wnt antagonists,Frizzled-4 / Fc or DKK1,was able to attenuated the Isl1 expression in P19CL6 cells,which could be modulated by the overexpression and knockdown of β-catenin. Ch IP assays demonstrated that the binding of LEF1 at- 2 300 bp upstream of Isl1 promoter was enhanced at day 4 of DMSO induction. The results suggested that canonical Wnt signaling to Isl1 expression during the early stage of cardiac differentiation was involved the binding of LEF1 / β-catenin to the Isl1 promoter.更多还原