力学信号耦联内胚层发育信号调控羊膜上皮细胞转分化为心肌细胞的实验研究

项目名称： 力学信号耦联内胚层发育信号调控羊膜上皮细胞转分化为心肌细胞的实验研究

项目编号： No.30870596

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 刘小菁

作者单位： 四川大学

项目金额： 32万元

中文摘要： 羊膜上皮细胞(amniotic epithelial cell, AEC)是心肌修复重建的理想种子细胞之一，但其转分化为心肌细胞的机理尚不清楚，而对内胚层发育信号以及心肌博动的力学刺激在调控AEC分化中作用的研究亦很少。本项目应用细胞生物力学、细胞生物学及分子生物学等方法技术观察了内胚层发育信号耦联心肌搏动的力学刺激诱导调控AEC转分化为心肌样细胞，并研究了这一过程中的分子机制。体外实验结果显示，通过应用内胚层END-2细胞的条件培养基培养7天，再辅以体外模拟心搏力学刺激的力学加载系统，经10％力学牵张刺激24小时的协同作用，可成功将AEC诱导为能表达心肌细胞特异转录因子和蛋白的心肌样细胞。Nkx-2.5、GATA-4、alpha-MHC等调控基因在此过程中发挥重要作用；BMP/Wnt/Nodal内胚层发育信号以及整合素-细胞骨架/ROS等力学信号途径参与此复杂的过程。进一步通过复制大鼠急性心肌梗死模型，观察到将AEC转分化的心肌样细胞移植入大鼠心肌梗死区后，能明显改善梗死大鼠的心功能。本项目研究成果对AEC分化及其在心血管疾病治疗的临床应用都将具有重要意义。

中文关键词： 力学刺激；内胚层发育信号；羊膜上皮细胞；转分化；心肌细胞

英文摘要： Amniotic epithelial cells (AECs) possess several advantages over embryonic and adult stem cells, and have been expected to be a good cell source for stem cell-based cardiac repair. However, efficient induction of AECs differentiation into cardiomyocytes remains a challenge. Signals arising from endoderm are essential to induce cardiogenesis in adjacent mesoderm, and mechanical stimulus is an effective technology for regenerative medicine. Therefore, the aim of present study is to investigate the synergistic effect of biochemical signals from endoderm and combined with mechanical stimuli on cardiomyocyte differentiation of AEC and the underlying mechanisms. AECs were isolated from human term placenta by trypsin digestion according to the previous reports. Freshly isolated hAECs were examined to detect the expression of epithelial cell marker cytokeratin 19, embryonic stem cell marker (SSEA-4) and stem cell-specific transcription factor (Oct-4). High-density undifferentiated hAECs at passage 1 were cultured with conditioned media from visceral-endoderm like cell line(the END-2 cells) for 7 days, and then subjected to 10% mechanical stretch and hypoxia(5% O2) for 24hours. After 7 days of culturing, mRNA and protein levels of cardiac-specific transcription factors (Nkx-2.5, GATA-4 and MEF-2C) and proteins, such as alpha- sarcomeric actinin, cardiac myosin heavy chain (alpha-MHC) and connexin-43, were significantly increased in treated AECs in response to conditional media from visceral endoderm cells, with further up-regulation when stimulated by combined use of 10% mechanical stretch for additional 24 hours. Several signals elicited by endoderm cells such as BMP/Wnt/Nodal and signals induced by mechanical stretch including cytoskeleton-integrin and ROS signals are involved in cardiomyocyte transdifferentiation of AEC. In vivo, acute myocardial infarction was created in adult male SD rats. The implantation of transdifferentiated AECs into infracted hearts of rats resulted in better heart functional recovery. Our results suggest that combined use of END-2 conditional media and mechanical stretch is synergistic for the cardiomyocyte transdifferentiation of hAECs, which might be a novel approach to induce transdifferentiation of AECs into cardiomyocytes. Taken together, this study might contribute to increase our knowledge of the mechanisms underlying transdifferentiation of AEC into cardiomyocyte-like cells and might be beneficial to stem cell therapy for cardiac repair.

英文关键词： Mechanical stimulation; Endoderm-derived developmental signals; Amniotic epithelial cells; Transdifferentiation; Cardiomyocyte