Nicotine Dysregulates Lung Differentiation through N-myc

描述(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。利用胚胎干细胞检测尼古丁对肺器官发生早期阶段的影响是一个独特的、正在出现的机会。这项建议使用定向分化方案中的胚胎干细胞来确定抑制N-myc水平是否是胚胎和胎儿肺发育期间尼古丁暴露的一种作用机制。我们的假设是，在体外，N-myc对人类胚胎干细胞向有功能的肺上皮和成纤维细胞的正常分化是必不可少的，尼古丁在体外和体内通过抑制N-myc信号通路阻止向肺的正常分化。在我们初步数据的基础上，这项建议将为吸烟母亲所生婴儿的肺功能下降提供一种机制，并将用于指导开发新的干预措施，以改善婴儿和儿童的肺健康。这一假设将通过两个特定的目标在翻译研究中得到验证：1)确定在人类胚胎干细胞定向分化为肺上皮和成纤维细胞的过程中，N-myc基因的敲除是否会导致基因表达模式的改变，增殖和凋亡率的失衡，以及无法在体外形成功能上皮。2)确定在向肺上皮和成纤维细胞分化的过程中，尼古丁是否导致N-myc表达降低，导致基因表达模式改变，细胞增殖和凋亡率异常，导致不能形成功能性上皮。这一目标也将确定在尼古丁存在的情况下，在分化过程中恢复N-myc表达是否可以防止尼古丁效应。尼古丁在体外的作用将通过小鼠体内肺发育的外植体模型来证实。

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.