基底刚度耦合机械拉伸促进肺泡上皮细胞分化的力学生物学机制研究

Pulmonary alveolar type I (AT1) cells are essential for gas exchage and one of the important components of alveolar. However, the differentiation of AT1 cells remains largely unknown. Although it has been reported that mechanical stimulation can induce the differentiation of AT1 from pulmonary alveolar type II (AT2) cells, its specific mechanical determinants and mechanisms have not been extensively studied. Based on the specially mechanical environment of lung, we propose to investigate the mechanical determinats and mechanisms of the differentiation of AT1 from AT2 cells by coupling substrate stiffness, cell spreading and mechanical stretch. We plan to accomplish the following tasks: fabricating geometric micropattern and micropost array with stiffness tunable in conjunction with microcontact printing and cell stretching device to achieve coupling substrate stiffness, cell spreading, and mechanical stretching to study their effects on the differentiation of AT1 from AT2 cells; based on the confocal microscopy platform, establishing a biomechanical experimental system for real-time dynamic observation of stem cell differentiation researches; precisely and quantitatively applying multi-factor mechanical stimulation, determining the biophysical and biochemical cues of microenvironment, and studying the molecular and biomechanical mechanisms in the differentiation of AT1 from AT2 cells. Approval and execution of the project as-proposed will definitely not only help our understanding of physiological and pathological mechanisms of lung regeneration, but also provide a new research platform for searching for the treatment of lung diseases.

I型肺泡上皮细胞（AT1）承担肺泡内气体交换功能，是肺泡结构的重要组成之一。然而AT1细胞的分化过程在很大程度上仍是未知。虽已有研究发现力学刺激可诱导II型肺泡上皮细胞（AT2）向AT1分化，但其力学调控因素与机制尚未得到广泛研究。本项目拟基于肺特异的力学环境，研究基底刚度、机械拉伸以及细胞铺展耦合作用对AT2向AT1分化的影响与力学调控机制。拟完成如下工作：制备几何微图案以及刚度可控的微柱阵列，并与微接触印刷及细胞拉伸装置结合，实现细胞铺展、基底刚度和机械拉伸的多因素耦合，研究其对AT2向AT1分化的影响；依托共聚焦显微镜，搭建用于实时动态观测干细胞分化的生物力学实验体系；精确定量施加多因素力学刺激，确定决定AT2向AT1分化的微环境理化因素、揭示其命运决定的分子及生物力学调控机制。本项目的实施也有望帮助深入了解肺再生的生理病理机制，以及为寻找肺疾病治疗方法提供新的研究平台。

本项目聚焦肺泡上皮细胞感知微环境刚度、几何约束及机械拉伸而分化进而参与肺发育与再生展开研究。构建了耦合细胞外基质刚度、几何限制、及机械拉伸的复杂微环境仿生系统，实现单或多因素力学与生化刺激，并初步确定决定肺上皮细胞表型转化及命运决定的微环境理化因素及力生物学机制。实验结果发现通过改变薄膜上微槽结构的特征尺寸能实现单轴和双轴的多模态应变场模式；发现机械拉伸耦合基质刚度调控肺泡上皮细胞命运决定，同时发现几何约束介导的肺泡上皮细胞片层内出现的异质性力学微环境就可诱发图案外围细胞自发发生表型转化，此过程受基质刚度调控；发现间质样与上皮样细胞间的力学传递因素在诱导触发上皮细胞群体迁移和浸润中起关键作用；发现荷包线结构介导的细胞-细胞定向收缩诱导机械异质性刚度梯度基底上上皮细胞层的群体迁移行为；发现微图案限制诱导的细胞多层空间堆叠可增强肠道球体的生物发生，揭示了新的用于控制组织形态发生的力学生物学范式。本项目的开展为研究肺组织发育再生及其他生物医学应用提供新工具，也为从力学生物学角度研究调控干细胞分化与命运决定奠定理论和实验基础。