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hESC的能力使科学家能够研究以前不可能的人胚胎分化机制。使用胚胎干细胞来检查尼古丁对肺器官形成的最早阶段的影响的能力是一个独特的新兴机会。该提案在定向分化方案中使用胚胎干细胞来确定N-myc水平的抑制是否是胚胎和胎儿肺发育期间尼古丁暴露的作用机制。我们的假设是N-myc暴露对于体外人胚胎干细胞正常分化为功能性肺上皮细胞和成纤维细胞是必不可少的，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，该提案将为吸烟母亲所生婴儿的肺功能下降提供一种机制，并用于指导新干预措施的开发，以改善婴儿和儿童的肺部健康。这一假设将在转化研究中使用两个具体目标进行测试：1）确定在定向分化为肺上皮和成纤维细胞期间敲低人胚胎干细胞中的N-myc是否导致基因表达模式改变、增殖和凋亡速率不平衡以及不能在体外形成功能性上皮。2)确定在分化为肺上皮和成纤维细胞期间暴露于尼古丁是否导致N-myc表达降低，从而导致基因表达模式改变，以及增殖和凋亡率异常，从而导致无法形成功能性上皮。这一目标还将确定在尼古丁存在下在分化期间恢复N-myc表达是否会防止烟碱效应。将使用体内肺发育的小鼠外植体模型证实尼古丁的体外作用。

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.