MSC肌分化在哮喘气道重塑肌层中的"修复"作用及调控

Airway remodeling is a key process and pathological characteristics of asthma pathogenesis, which the source of smooth muscular layer cells is not clear. Many reports and our previous study found muscle layer cells possess mesenchymal stem cells (MSC) characteristics in the airways of asthma mice and there were ASMC appeared on respiratory bronchioles where was considered as no ASMC of asthma mice and take along molecular phenotypic characteristics of MSC. We hypothesized that MSC is likely to be precursor cells of abnormally growth asthma ASMC and participate in the airway remodeling. This project intends to use CreERt2/Loxp specific locus gene reorganization system to construct genetically engineered mice that could be regulated on gene levels of ASMC and MSC with special fluorescence. We study cell migration, proliferation and differentiation process of model mice both ASMC and MSC through genetic lineage tracer combined with the laser imaging of. Besides, we analyze the ultrastructure, contraction and secretion of ASMC cells that derived from MSC and the own ASMC with transmission electron microscopy. With conditionally remove MSC in iDRT genetically engineered mice to discuss the effect on lack of MSC in asthma airway remodeling. With the gene knockout technology to reveal the regulation function of TGF-beta/Smad signal pathways on MSC myogenic differentiation and provide thought for asthma therapy.

气道重塑是哮喘发病的关键环节及病理特征，但增厚的平滑肌层细胞来源及发生机制并不清楚。文献报道哮喘小鼠气道肌层细胞具有间充质干细胞（MSC）部分特征，我们前期亦发现，哮喘小鼠本无气道平滑肌细胞（ASMC）的呼吸性细支气管却出现了兼具ASMC和MSC分子特征的细胞。我们推测，哮喘气道重塑增厚肌层的这类“ASMC”可能是气道重塑修复过程中MSC肌分化的结果。本课题拟用CreERt2/Loxp系统，构建携带荧光的ASMC及MSC工程小鼠；遗传谱系示踪模型小鼠ASMC及MSC的迁移、增殖、分化，明确MSC来源的ASMC在重塑肌层中的占比；采用免疫透射电镜等对比分析气道重塑肌层中自身ASMC来源及MSC来源的ASMC超微结构及收缩、分泌功能；利用iDRT小鼠条件性剔除技术探讨MSC缺失对哮喘气道重塑的影响；采用基因敲除技术探讨TGF-β/Smad信号通路对MSC肌分化的调控，为哮喘治疗提供思路。

气道重塑是哮喘发病的关键环节及病理特征之一，但增厚的平滑肌层细胞来源及发生机制并不清楚。本研究构建了Gli1+MSC-eYFP和MYH11+ASMC-tdTomato谱系遗传示踪小鼠，发现Gli1+MSC可肌分化肌成纤维细胞，参与上皮下纤维化，并由TGF-β和Wnt信号通路共同调控。还发现具有平滑肌细胞分子标志物的自身来源的MYH11+ASMC参与哮喘气道肌层重塑，同时也发现气道重塑肌层部分具有平滑肌细胞分子标志物的α-SMA阳性的细胞并非来源于自身气道平滑肌细胞增殖的“ASMC”。通过scRNA-seq和细胞分化轨迹联合分析，发现Sca1+MSC、Nestin+MSC和PDGFRα+MSC可肌分化为气道平滑肌细胞，参与哮喘气道肌层重塑。通过对哮喘肺组织中MSC和MSC来源的“ASMC”细胞差异基因进行分析，发现CSRP1可通过与SRF相结合调控MSC肌分化，参与哮喘气道重塑。此外，scRNA-seq和scATAC-seq联合分析亦发现，肌成纤维细胞可转分化为ASMC参与哮喘气道重塑。上述研究结果进一步完善了哮喘气道重塑肌层细胞来源的基础理论，同时为靶向哮喘气道重塑的治疗提供新靶点。