间充质干细胞介导的线粒体转移对修复氧化应激引起气道细胞损伤的机制性研究

Mesenchymal stem cells (MSCs) administration is a promising clinical strategy to treat patients suffering from oxidative stress-dependent lung diseases such as chronic obstructive pulmonary disease (COPD). Our previous work reported on mitochondrial transfer via tunneling nanotubes (TNT) from induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) to oxidative stress-mediated bronchial epithelial or smooth muscle cell injury. However, the elucidation of the underlying mechanism and pathophysiological significance of this transfer requires further study. Kinesin superfamily proteins (Kifs) are microtubule-dependent molecular motor proteins that transport organelles and protein complexes in cells. There are 3 isoforms of Kif5s of kinesin heavy chains exist in mammals; Kif5a and Kif5c are mainly expressed in neurons, whereas Kif5b is expressed ubiquitously. In this proposal, we hypothesize that mitochondrial movement will depends on microtubules and the specific motor protein. We aim to determine the movement velocity of mitochondria in TNT of MSC-mediated mitochondrial transfer using microtubule polymerization inhibitors nocodazole or cocemid, and knockdown of the microtubule motor protein kinesin family member 5b (Kif5b). We will first compare naïve MSCs with Kif5b(hi) MSCs in mitochondrial transfer to cigarette smoke-mediated airway cell (epithelial and smooth muscle cells) injury and functional recovery of mitochondria through bioenergetics determination. We will then administer naïve MSCs and Kif5b(hi) MSCs into cigarette smoke (CS)- or ozone-exposed rodent models to find the efficacy of stem cells in rescuing oxidative stress-induced chronic lung injury and pulmonary dysfunction. We will also examine mitochondrial transfer and functions as well as tissue remodeling processes using immunocytochemistry of live imaging. The levels and subcellular localization of Kifs will be examined by RT-PCR, Western blot analysis and immunocytochemistry. Understanding the mechanisms of Kif5b regulation in MSCs will facilitate the development of high efficacy MSC-based therapeutic approach to effectively treat oxidative stress-induced lung injuries as in COPD.

间充质干细胞（MSCs）具有广泛的临床应用前景。基于本课题组前期研究，诱导性多功能干细胞来源的MSCs（iPSC-MSCs）通过依赖纳米管实现线粒体转移，从而对氧化应激引起气道细胞损伤起修复作用。但是，线粒体转移的具体机制及其生理学意义需要进一步的深入研究。本项目中，我们假设间充质干细胞介导的线粒体转移取决于微管和特定的马达蛋白。我们拟通过比较iPSC-MSCs和不同Kif5b表达的iPSC-MSCs在香烟烟雾引起气道细胞损伤模型中对线粒体转移和功能恢复的影响；并应用这些干细胞于香烟烟雾或臭氧暴露动物模型中，以确定干细胞舒缓肺部损伤和恢复肺功能的有效性；利用活细胞追踪技术，实时观测线粒体的运动与运输；检测Kifs的表达水平和亚细胞定位，最终探究Kif5b的调控机制。本研究的结果，将有助于开发高效干细胞的治疗手段，为实现干细胞治疗氧化应激引起的肺部疾病（如慢性阻塞性肺病）奠定基础。

为研究气道上皮细胞中的线粒体转移， 我们建立了模拟人气道上皮的气液界面 （air-liquid interface，ALI）培养模型， 在该模型中， 细胞的顶端暴露于空气中， 营养物质提供于细胞的基底外侧。 经过28天的培养， 原代人支气管上皮细胞 （human bronchial epithelial cells， HBEpCs ）完全分化， 并形成典型的假复层气道上皮，呈现出细胞间紧密连接， 并分化出MUC5AC阳性染色的杯状细胞且α-tubulin 阳性染色的纤毛细胞。 该模型培养的细胞高度模拟了导气管表面上皮的形态与功能。 将细胞表面暴露于香烟烟雾介质 （cigarette smoke medium， CSM, 4%）后， 细胞死亡数量显著增加， 且通过MUC5AC染色观察到杯状细胞数量显著增加，表明HBEpCs受到损伤。 因此我们假设， 上述气道上皮培养条件可能有助于测定间充质干细胞 （mesenchymal stem cells，MSCs）向HBEpCs的移动。 为了验证假设，ALI培养的原代HBEpCs与转染上mito-GFP用于标记MSC来源的线粒体的诱导多能干细胞衍生间充质干细胞 （induced pluripotent stem cell-derived mesenchymal stem cells，iPSC-MSCs）进行共培养。 通过实时捕获，我们通过活细胞成像显微镜发现MSCs从分化细胞的顶端向基底外侧移动。 吸烟是慢性阻塞性肺病 (COPD) 患者的主要危险因素， 因此， 本研究建立的体外共培养系统为选择iPSC-MSCs群体提供了非常有价值的方法， 通过筛选出具有更强线粒体转移能力的iPSC-MSCs用以修复COPD患者受损的气道上皮。