Core-003

核心003

• 批准号: 10477256
• 负责人: John Paul Clancy
• 金额: $ 22.93万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10477256
• 关键词: Air; Atlases; Behavior; Bioinformatics; Cell Culture Techniques; Cell Differentiation process; Cell Separation; Cells; Characteristics; Chromium; Code; Communities; Confocal Microscopy; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA sequencing; Data; Data Set; Electrolytes; Epithelial Cells; Evaluation; Fibroblasts; Fluorescence Microscopy; Gene Expression; Generations; Genotype; Goblet Cells; Growth; Immunofluorescence Immunologic; In Vitro; Individual; Ion Transport; Liquid substance; Lung; Methodology; Microfluidics; Molecular; Morphology; Mucous body substance; Mus; Mutation; Nasal Epithelium; Nose; Patients; Phenotype; RNA Databases; RNA analysis; Research; Research Personnel; Rho-associated kinase; Series; Serous; Therapeutic Studies; Untranslated RNA; Western Blotting; airway epithelium; airway surface liquid; bioinformatics pipeline; bronchial epithelium; cell type; droplet sequencing; epithelial Na+ channel; experimental study; in vitro Model; individualized medicine; kinase inhibitor; molecular imaging; monolayer; single-cell RNA sequencing; transcriptome; transcriptome sequencing; transcriptomics; web site; whole genome

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.

“CF呼吸道上皮细胞的功能和转录组图谱”旨在建立一个核心， 分离和扩增呼吸道上皮细胞从患者的鼻和支气管刷 特异性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将在 在单细胞水平上研究。共聚焦显微镜和免疫荧光将被用来仔细识别 CFTR突变和“校正剂”对细胞分化、表型和功能的影响。详细 将使用单细胞RNA-seq产生鼻和支气管细胞培养物的细胞特异性RNA数据库。 详细的分子成像，通过共聚焦显微镜，将被用来创建一个基因表达图谱， 与CF有关的行为CFTR校正剂对特定患者突变的影响将在这些研究中进行探索。 体外模型 具体目标1将分离、培养和评价呼吸道上皮细胞，用于研究支气管CF 刷牙Specific Aim 2将使用NexGen单细胞RNA测序来创建患者特异性 呼吸道上皮细胞转录组学这些研究将确定CFTR突变对基因表达的影响。 在单细胞水平上的表达，鉴定CFTR突变和校正剂对行为的影响， 多种细胞类型的单个细胞，包括纤毛细胞，浆液细胞和杯状细胞，将出现在这些细胞中。 cultures.核心将创建一个分子图谱，它将是细胞特异性的，患者特异性的，突变特异性的， 提供广泛的数据集，供全球CF研究界的研究人员使用。等 数据将使我们能够理解CFTR、CFTR突变和CFTR的多效性效应。 矫正器以患者特异性的方式，因为我们朝着囊性纤维化的患者特异性疗法发展。