Core-003

核心003

基本信息

批准号：
10224484
负责人：
John Paul Clancy
金额：
$ 23.36万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
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/10224484
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 Structure of respiratory epithelium 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

项目摘要

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。将在培养物中评估 CFTR 突变对细胞特异性转录组的影响。将使用共聚焦显微镜和免疫荧光在单细胞水平上进行仔细识别。 CFTR 突变和“校正因子”对细胞分化、表型和功能的影响。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 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 Specific Aim 2 将使用 NexGen 单细胞 RNA 测序来创建患者特异性图谱。这些研究将确定 CFTR 突变对基因的影响。单细胞水平的表达，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核心将创建一个分子图谱，该图谱将针对细胞、患者和突变。提供广泛的数据集，供活跃于全球 CF 研究界的研究人员使用。数据将使我们能够了解 CFTR、CFTR 突变和 CFTR 的多效性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。