Core-003

“CF 呼吸道上皮细胞功能和转录组图谱”旨在建立一个核心 从患有以下疾病的患者的鼻腔和支气管刷检中分离并扩增呼吸道上皮细胞 特定的 CFTR 突变。细胞将在支气管球和鼻球中培养。液体和电解质 将在培养物中评估分泌。 CFTR 突变对细胞特异性转录组的影响 在单细胞水平上进行研究。将使用共聚焦显微镜和免疫荧光来仔细识别 CFTR 突变和“校正子”对细胞分化、表型和功能的影响。详细 将使用单细胞 RNA-seq 为鼻和支气管细胞培养物生成细胞特异性 RNA 数据库。 通过共聚焦显微镜进行的详细分子成像将用于创建基因表达和基因图谱 与 CF 相关的行为。 CFTR 校正因子对特定患者突变的影响将在这些中进行探讨 体外模型。 具体目标 1 将分离、培养和评估呼吸道上皮细胞，以研究支气管 CF 刷牙。具体目标 2 将使用 NexGen 单细胞 RNA 测序来创建患者特异性图谱 呼吸道上皮细胞转录组学。这些研究将确定 CFTR 突变对基因的影响 在单细胞水平上表达，确定 CFTR 突变和校正因子对行为的影响 多种细胞类型的单个细胞，包括存在于这些细胞中的纤毛细胞、浆液细胞和杯状细胞 文化。核心将创建一个分子图谱，该图谱将针对细胞、患者和突变，并将 提供广泛的数据集，供活跃在全球 CF 研究界的研究人员使用。这样的 数据将使我们能够了解 CFTR、CFTR 突变和 CFTR 的多效性效应 随着我们朝着针对囊性纤维化的患者特定疗法迈进，以患者特定的方式进行校正。

The “Functional and Transcriptomic Atlas of CF Respiratory Epithelial Cells” seeks to establish a Core to isolate and expand respiratory epithelial cells from both nasal and bronchial brushings from patients with specific CFTR mutations. Cells will be cultured in bronchospheres and nasospheres. Fluid and electrolyte secretion will be assessed in the cultures. Effects of CFTR mutations on cell-specific transcriptomes will be studied at single cell level. Confocal microscopy and immunofluorescence will be used to carefully identify the effects of CFTR mutations and “correctors” on cell differentiation, phenotypes, and functions. Detailed cell-specific RNA databases will be produced for nasal and bronchial cell cultures using single cell RNA-seq. Detailed molecular imaging, by confocal microscopy, will be used to create an Atlas of gene expression and behavior related to CF. Effects of CFTR correctors on specific patient mutations will be explored in these in vitro models. Specific Aim 1 will isolate, culture, and evaluate respiratory epithelial cells for study of CF from bronchial brushings. Specific Aim 2 will use NexGen single cell RNA-sequencing to create an Atlas of patient-specific respiratory epithelial cell transcriptomics. These studies will identify the effects of CFTR mutations on gene expression at the single cell level, identifying the effects of CFTR mutations and correctors on the behaviors of individual cells of multiple cell types, including ciliated, serous, and goblet cells that will be present in these cultures. The Core will create a molecular Atlas which will be cell-, patient-, and mutation-specific and will provide an extensive data set for use by investigators active in the CF research community worldwide. Such data will enable the ability to understand the pleotropic effects of CFTR, CFTR mutations, and CFTR correctors in a patient-specific manner, as we move toward patient-specific therapies for cystic fibrosis.