Mechanisms and Targeted Therapy of Airway Basal Cell Dysfunction in Bronchiolitis Obliterans Syndrome

Haikou Affiliated Hospital of Central South University Xiangya School of Medicine

Status: Invitation-only

Conditions: Bronchiolitis Obliterans Syndrome (BOS)

Treatments: Procedure: Bronchoscopic brushing; Genetic: Cell function identification; Genetic: Identification of local microenvironmental changes; Procedure: Prevention of ferret airway basal cell transplantation; Genetic: Single - cell cloning; Procedure: Transplantation treatment of ferret airway basal cells; Genetic: Gene editing

Study type: Interventional

Funder types: Other

Identifiers

NCT07018804

SC20250160

Details and patient eligibility

About

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.

Full description

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.

Enrollment: 5 estimated patients

Sex: All

Volunteers: Accepts Healthy Volunteers

Inclusion criteria

• Patients who have received allogeneic hematopoietic stem cell transplantation and are diagnosed with bronchiolitis obliterans syndrome

Exclusion criteria

• Bronchoscopy consultation is not suitable for patients who are not suitable for bronchoscopy.

Trial design

Primary purpose: Supportive Care

Allocation: Non-Randomized

Interventional model: Parallel Assignment

Masking: None (Open label)

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5 participants in 8 patient groups

Patient group

Experimental group

Description:

Epithelial mucosal tissues from the diseased lung regions and relatively healthy lung lobes of BOS patients following hematopoietic stem cell transplantation (HSCT) will be collected. Alveolar lavage fluid specimens will be obtained when available. The intervention consists of digesting the collected samples with tissue collagenase for the culture of airway basal cells (BCs).

Treatment:

Procedure: Bronchoscopic brushing

Healthy control group

Experimental group

Description:

Healthy volunteers with no obvious airway abnormalities will be selected to collect samples from the same anatomical sites. No disease-related interventions will be performed, serving as a comparative control to highlight the abnormal conditions of basal cells (BCs) in the patient group.

Treatment:

Procedure: Bronchoscopic brushing

Single - cell clone group

Experimental group

Treatment:

Genetic: Single - cell cloning

Cell Function Identification Group

Experimental group

Treatment:

Genetic: Cell function identification

Local microenvironment change identification group

Experimental group

Treatment:

Genetic: Identification of local microenvironmental changes

Gene Editing Group

Experimental group

Treatment:

Genetic: Gene editing

Ferret airway basal cell transplantation group (prevention group)

Experimental group

Treatment:

Procedure: Prevention of ferret airway basal cell transplantation

Ferret airway basal cell transplantation group (treatment group)

Experimental group

Treatment:

Procedure: Transplantation treatment of ferret airway basal cells