Nicotine Dysregulates Lung Differentiation through N-myc

描述（由申请人提供）：在定向分化方案中使用hESCs的能力使科学家能够研究人类胚胎分化的机制，这在以前是不可能的。利用胚胎干细胞来检测尼古丁对肺器官发生早期阶段的影响是一个独特而新兴的机会。本研究利用胚胎干细胞进行定向分化，以确定N-myc水平的抑制是否是胚胎和胎儿肺发育过程中尼古丁暴露的作用机制。我们的假设是，暴露于N-myc对于人胚胎干细胞在体外正常分化为功能性肺上皮细胞和成纤维细胞是必要的，尼古丁通过抑制N-myc信号通路阻止体外和体内正常分化为肺。基于我们的初步数据，这一建议将提供吸烟母亲所生婴儿肺功能下降的机制，并用于指导开发新的干预措施，以改善婴儿和儿童的肺部健康。这一假设将在转化研究中进行验证，目的有两个：1)确定在定向分化为肺上皮细胞和成纤维细胞的过程中，人胚胎干细胞中N-myc的敲除是否会导致基因表达模式改变、增殖和凋亡率失衡，以及在体外无法形成功能性上皮。2)确定在肺上皮和成纤维细胞分化过程中暴露于尼古丁是否导致N-myc表达降低，导致基因表达模式改变，增殖和凋亡率异常，导致不能形成功能性上皮。这一目的还将确定在尼古丁存在的情况下恢复分化过程中的N-myc表达是否能防止尼古丁的影响。尼古丁的体外作用将通过小鼠体内肺发育的外植体模型得到证实。

DESCRIPTION (provided by applicant): The ability to use hESCs in directed differentiation protocols allows scientists to investigate mechanisms of human embryonic differentiation that were not previously possible. The ability to use embryonic stem cells to examine the effect of nicotine on the earliest stages of lung organogenesis is a unique and emerging opportunity. This proposal uses embryonic stem cells in directed differentiation protocols to determine if suppression of N-myc levels is a mechanism of action for nicotine exposure during embryonic and fetal lung development. Our hypothesis is that N-myc exposure is essential for normal differentiation of human embryonic stem cells into functional lung epithelium and fibroblasts in vitro, and that nicotine prevents normal differentiation into lung in vitro and in vivo by inhibiting the N-myc signaling pathway. Building on our preliminary data, this proposal will provide a mechanism for the decreased lung function seen in infants born to smoking mothers, and be used to guide the development of new interventions to improve the lung health of infants and children. This hypothesis will be tested in translational research using two specific aims: 1) To determine if knock-down of N-myc in human embryonic stem cells during directed differentiation into lung epithelium and fibroblasts leads to altered gene expression patterns, imbalanced rates of proliferation and apoptosis, and an inability to form a functional epithelium in vitro. 2) To determine if exposure to nicotine during differentiation into lung epithelium and fibroblasts leads to decreased N-myc expression, resulting in altered gene expression patterns, and abnormal rates of proliferation and apoptosis, resulting in the inability to form a functional epithelium. This aim will also determine if restoring N-myc expression during differentiation in the presence of nicotine prevents nicotinic effects. Effects of nicotine in vitro will be confirmed using a murine explant model of in vivo lung development.