Mechanisms and Targeted Therapy of Airway Basal Cell Dysfunction in Bronchiolitis Obliterans Syndrome
NCT ID: NCT07018804
Last Updated: 2025-06-12
Study Results
The study team has not published outcome measurements, participant flow, or safety data for this trial yet. Check back later for updates.
Basic Information
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ENROLLING_BY_INVITATION
NA
5 participants
INTERVENTIONAL
2026-01-01
2028-06-30
Brief Summary
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Detailed Description
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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.
Conditions
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Study Design
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NON_RANDOMIZED
PARALLEL
SUPPORTIVE_CARE
NONE
Study Groups
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Patient group
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).
Bronchoscopic brushing
The collected samples are digested with tissue collagenase for the culture of airway basal cells (BCs).
Healthy control group
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.
Bronchoscopic brushing
The collected samples are digested with tissue collagenase for the culture of airway basal cells (BCs).
Single - cell clone group
Single - cell cloning
After primary BCs are expanded in the P1 passage, single-cell cloning libraries are established by planting them into 384-well cell culture plates in a single-cell per well format using a flow chamber cell sorter. Ten samples are selected from the expandable clones for the identification of differentiation and proliferation capabilities, while the remaining clones are cryopreserved.
Cell Function Identification Group
Cell function identification
For single-cell samples of each patient, in vitro expansion culture is performed to observe the morphology of cells at each passage and calculate the clonogenic rate. Immunofluorescence technique is used to detect the expression of the cell proliferation marker Ki67, and the proliferation capacity is evaluated by combining with the CCK8 assay. Cells at passages P3-P5 are seeded on the permeable membrane of cell culture inserts at a density of \\(10\^6\\) cells/cm², and after 21 days of culture, the differentiated structures are collected. The expression of ciliated cell marker Ace-Tubulin and goblet cell marker MUC5AC is detected to assess the differentiation capacity. Meanwhile, cells are injected subcutaneously into NSG mice at \\(10\^6\\) cells/injection site for in vivo differentiation for 28 days, and the differentiated structures are collected for pathological analysis.
Local microenvironment change identification group
Identification of local microenvironmental changes
The air-liquid interface (ALI) differentiation culture medium is collected, and cell debris is removed by high-speed centrifugation. The supernatant is then collected to extract proteins. Target proteins are purified via immunoprecipitation or affinity chromatography, followed by desalting and concentration for mass spectrometry analysis to detect inflammatory cytokines and extracellular matrix (ECM). Real-time PCR and Western blot techniques are used to measure the expression levels of epithelial markers, mesenchymal markers, and ECM in cells across all groups, thereby evaluating changes in the local microenvironment around small airways.
Gene Editing Group
Gene editing
After plasmid construction, based on gene function, the CRISPR-Cas9-sgRNA (all-in-one) plasmid is transiently transfected into expanded single-cell strains via Nucleofection to knockout the target gene, or the CRISPR-dCas9 fusion-sgRNA plasmid is transiently transfected to activate or inhibit the target gene. After 3-5 days of culture, viable cells are sorted by FACS, and single cells are seeded into 96-well plates for two additional passages of expansion. Samples are collected for Sanger sequencing to screen successfully constructed cell lines.
Ferret airway basal cell transplantation group (prevention group)
Prevention of ferret airway basal cell transplantation
Before surgery, basal cells (BCs) of recipient ferrets are collected by bronchoscopic brushing, followed by in vitro culture and identification. Prior to the onset of BOS, the cells are injected into recipient ferrets via bronchoscopy. Outcomes including ferret survival rate, body weight, CT imaging, and lung tissue pathology are collected to evaluate the preventive effect of BCs on BOS.
Ferret airway basal cell transplantation group (treatment group)
Transplantation treatment of ferret airway basal cells
Stable cell lines with target gene knockdown are established in ferret basal cells (BCs). After ferrets develop bronchiolitis obliterans syndrome (BOS), the constructed cell lines are transplanted into recipient ferrets to validate the therapeutic effect of gene-corrected BCs on the disease.
Interventions
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Bronchoscopic brushing
The collected samples are digested with tissue collagenase for the culture of airway basal cells (BCs).
Single - cell cloning
After primary BCs are expanded in the P1 passage, single-cell cloning libraries are established by planting them into 384-well cell culture plates in a single-cell per well format using a flow chamber cell sorter. Ten samples are selected from the expandable clones for the identification of differentiation and proliferation capabilities, while the remaining clones are cryopreserved.
Cell function identification
For single-cell samples of each patient, in vitro expansion culture is performed to observe the morphology of cells at each passage and calculate the clonogenic rate. Immunofluorescence technique is used to detect the expression of the cell proliferation marker Ki67, and the proliferation capacity is evaluated by combining with the CCK8 assay. Cells at passages P3-P5 are seeded on the permeable membrane of cell culture inserts at a density of \\(10\^6\\) cells/cm², and after 21 days of culture, the differentiated structures are collected. The expression of ciliated cell marker Ace-Tubulin and goblet cell marker MUC5AC is detected to assess the differentiation capacity. Meanwhile, cells are injected subcutaneously into NSG mice at \\(10\^6\\) cells/injection site for in vivo differentiation for 28 days, and the differentiated structures are collected for pathological analysis.
Identification of local microenvironmental changes
The air-liquid interface (ALI) differentiation culture medium is collected, and cell debris is removed by high-speed centrifugation. The supernatant is then collected to extract proteins. Target proteins are purified via immunoprecipitation or affinity chromatography, followed by desalting and concentration for mass spectrometry analysis to detect inflammatory cytokines and extracellular matrix (ECM). Real-time PCR and Western blot techniques are used to measure the expression levels of epithelial markers, mesenchymal markers, and ECM in cells across all groups, thereby evaluating changes in the local microenvironment around small airways.
Gene editing
After plasmid construction, based on gene function, the CRISPR-Cas9-sgRNA (all-in-one) plasmid is transiently transfected into expanded single-cell strains via Nucleofection to knockout the target gene, or the CRISPR-dCas9 fusion-sgRNA plasmid is transiently transfected to activate or inhibit the target gene. After 3-5 days of culture, viable cells are sorted by FACS, and single cells are seeded into 96-well plates for two additional passages of expansion. Samples are collected for Sanger sequencing to screen successfully constructed cell lines.
Prevention of ferret airway basal cell transplantation
Before surgery, basal cells (BCs) of recipient ferrets are collected by bronchoscopic brushing, followed by in vitro culture and identification. Prior to the onset of BOS, the cells are injected into recipient ferrets via bronchoscopy. Outcomes including ferret survival rate, body weight, CT imaging, and lung tissue pathology are collected to evaluate the preventive effect of BCs on BOS.
Transplantation treatment of ferret airway basal cells
Stable cell lines with target gene knockdown are established in ferret basal cells (BCs). After ferrets develop bronchiolitis obliterans syndrome (BOS), the constructed cell lines are transplanted into recipient ferrets to validate the therapeutic effect of gene-corrected BCs on the disease.
Eligibility Criteria
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Inclusion Criteria
Exclusion Criteria
ALL
Yes
Sponsors
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Haikou Affiliated Hospital of Central South University Xiangya School of Medicine
OTHER
Responsible Party
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Principal Investigators
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Xiaoyang Yang, DM
Role: PRINCIPAL_INVESTIGATOR
Department of Hematology, Haikou People's Hospital
Locations
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Haikou Affiliated Hospital of Central South University Xiangya School of Medicine
Haikou, Hainan, China
Countries
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Other Identifiers
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SC20250160
Identifier Type: -
Identifier Source: org_study_id
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