Analysis of the Pathogenic Mechanism of Abnormal Airway Basal Cell Function in Bronchiolitis Obliterans Syndrome and Research on Targeted Treatment Strategies
Analysis of the Pathogenic Mechanism of Abnormal Airway Basal Cell Function in Bronchiolitis Obliterans Syndrome and Research on Targeted Treatment Strategies
This experimental study aims to investigate the pathogenesis of bronchiolitis obliterans syndrome (BOS) and provide a basis for clinical diagnosis and treatment. The core research question is: whether there is a causal relationship between stem cell dysfunction induced by the inflammatory microenvironment and airway injury repair during the pathological process of BOS? Researchers will collect alveolar lavage fluid specimens from participants and healthy individuals to isolate distal small airway stem cells for subsequent scientific research and comparative analysis, thereby revealing the pathological mechanisms of BOS, exploring precise intervention targets, and developing innovative therapeutic strategies to improve patient prognosis, long-term survival rates, and quality of life.
The purpose of this experimental study is to explore the role of airway basal cells in the development of bronchiolitis obliterans syndrome (BOS) and develop potential therapeutic strategies. The main question it aims to answer is: How do the functional abnormalities of airway basal cells (BCs) affect the progression of BOS and what are the underlying molecular mechanisms? Researchers will collect epithelial mucosal tissues from diseased and relatively healthy lung regions of BOS patients through bronchoscopic brushing, and simultaneously gather alveolar lavage fluid specimens when possible. Specimens from healthy volunteers with no obvious airway abnormalities will be used as controls. After that, BCs will be isolated from these specimens. The isolated BCs will be cultured and their molecular characteristics will be identified using immunofluorescence staining to detect the expression of BCs markers such as p63 and Krt5.
Subsequently, single - cell clone libraries will be established by flow cytometry cell fluorescence sorting (FACS) technology. Multiple aspects of the cells' functions will be evaluated, including self - renewal ability by detecting the expression of the proliferation marker Ki67 through immunofluorescence and CCK8 assay, and differentiation ability by analyzing the cell types and proportions in the differentiation structures of in vitro air - liquid interface (ALI) culture for 21 days and in vivo differentiation in severe combined immunodeficiency (NSG) mice for 28 days using Real - time PCR and immunofluorescence techniques.
In addition, multi - omics sequencing technologies, such as RNA - seq, ATAC - seq, and CUT&Tag, will be employed to explore the molecular mechanisms of BCs' functional abnormalities. Stable interference cell lines will be established using CRISPR - Cas9 technology to verify the functions of potential target genes. A ferret BOS model will be constructed, and BCs transplantation experiments will be carried out in ferrets. By observing and analyzing the survival rate, body weight, CT images, and lung tissue pathology of ferrets, the preventive and therapeutic effects of BCs on BOS will be evaluated.
By clarifying the role of BCs in BOS, this study aims to reveal the underlying pathological mechanisms, explore potential intervention targets, and develop novel treatment approaches. These efforts are expected to improve the prognosis of BOS patients, reduce the incidence and mortality rates, and enhance the overall quality of life for those affected by this life - threatening respiratory disorder.
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