Study was withdrawn prior to enrollment due to dissolution of collaborative partnership. The protocol may be reactivated in the future under new collaboration.
Analysis of the Pathogenic Mechanism of Abnormal Airway Basal Cell Function in Bronchiolitis Obliterans Syndrome and Research on Targeted Treatment Strategies
Sponsor study identifier
interventional
0
1 country
1
Study Start
July 26, 2026
Estimated (date passed)This estimate has passed. The registry has not confirmed an actual date for this milestone.
Primary Completion
June 30, 2027
EstimatedPlanned date reported by the registry; this milestone is not confirmed as achieved.
Study Completion
June 30, 2028
EstimatedPlanned date reported by the registry; this milestone is not confirmed as achieved.
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.
Registry-reported information. Elapsed time and enrollment totals alone do not measure study progress or recruitment pace.
June 30, 2028
EstimatedPlanned date reported by the registry; this milestone is not confirmed as achieved.
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Dates and qualifiers reported by the registry. Estimated dates do not confirm that a milestone has occurred.
May 17, 2025
Actual (reported)June 12, 2025
Actual (reported)July 26, 2026
Estimated (date passed)This estimate has passed. The registry has not confirmed an actual date for this milestone.
June 30, 2027
EstimatedPlanned date reported by the registry; this milestone is not confirmed as achieved.
June 30, 2028
EstimatedPlanned date reported by the registry; this milestone is not confirmed as achieved.
Ki67 Expression Level in Airway Basal Cells
The percentage of Ki67-positive airway basal cells assessed by immunohistochemistry. Ki67 is a nuclear protein associated with cellular proliferation. Higher Ki67 expression indicates greater proliferative activity. Comparison will be made between patients with bronchiolitis obliterans syndrome and healthy controls to evaluate abnormal self-renewal function of airway basal cells in disease states.
From enrollment to 32 weeks
Cell Proliferative Activity Assessed by CCK8 Assay
Optical density (OD) values measured by the CCK8 (Cell Counting Kit-8) assay in cultured airway basal cells. The CCK8 assay measures cell viability and proliferation based on the reduction of WST-8 by dehydrogenases in living cells. Higher OD values indicate greater cell proliferation. Comparison will be made between patients with bronchiolitis obliterans syndrome and healthy controls.
From enrollment to 32 weeks
Expression of Ciliated Cell Marker Ace-Tubulin in Air-Liquid Interface (ALI) Differentiation Culture
Immunofluorescence staining intensity of Ace-Tubulin (acetylated α-tubulin), a marker for ciliated cells, in airway basal cells cultured under air-liquid interface (ALI) conditions for 21 days. The staining intensity reflects the degree of ciliated cell differentiation from airway basal cells.
From enrollment to 32 weeks
Expression of Goblet Cell Marker MUC5AC in Air-Liquid Interface (ALI) Differentiation Culture
Immunofluorescence staining intensity of MUC5AC, a marker for goblet cells, in airway basal cells cultured under air-liquid interface (ALI) conditions for 21 days. The staining intensity reflects the degree of goblet cell differentiation from airway basal cells.
Expression of Airway Basal Cell Markers p63 and Krt5 by Immunofluorescence
From enrollment to 32 weeks
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).
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.
The collected samples are digested with tissue collagenase for the culture of airway basal cells (BCs).
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.
Haikou Affiliated Hospital of Central South University Xiangya School of Medicine
Haikou, Hainan, 570208, China
Xiaoyang Yang, DM
Department of Hematology, Haikou People's Hospital
0 participants (actual enrollment reported by the registry).
First Submitted
May 17, 2025
First Posted
June 12, 2025
ex Vivo Study of Liposomes Loaded With Everolimus in Chronic Lung Allograft Disfunction
From enrollment to 32 weeks
Expression of Ciliated Cell Marker Ace-Tubulin in NSG Mouse Xenograft Model
Immunofluorescence staining intensity of Ace-Tubulin (acetylated α-tubulin), a marker for ciliated cells, in airway basal cells differentiated in vivo in severe combined immunodeficiency (NSG) mice for 28 days.
From enrollment to 32 weeks
Expression of Goblet Cell Marker MUC5AC in NSG Mouse Xenograft Model
Immunofluorescence staining intensity of MUC5AC, a marker for goblet cells, in airway basal cells differentiated in vivo in severe combined immunodeficiency (NSG) mice for 28 days.
From enrollment to 32 weeks
Survival Rate of Ferrets After Airway Basal Cell Transplantation
Percentage of ferrets surviving at the end of the 32-week observation period following airway basal cell transplantation. Survival is recorded daily. This indicator evaluates the impact of airway basal cell transplantation on disease progression in the bronchiolitis obliterans syndrome ferret model.
From enrollment to 32 weeks
Body Weight Changes in Ferrets After Airway Basal Cell Transplantation
Body weight of ferrets measured at baseline and at regular intervals (weekly) over the 32-week observation period following airway basal cell transplantation. Weight change is calculated as the percentage change from baseline. This indicator reflects the overall health status of ferrets and assists in evaluating the effect of airway basal cell transplantation.
From enrollment to 32 weeks
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.
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.
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.
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.
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.
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Study Start
July 26, 2026
Estimated (date passed)This estimate has passed. The registry has not confirmed an actual date for this milestone.
Primary Completion
June 30, 2027
EstimatedPlanned date reported by the registry; this milestone is not confirmed as achieved.
Study Completion
June 30, 2028
EstimatedPlanned date reported by the registry; this milestone is not confirmed as achieved.
Last Updated
July 29, 2026
Record last verified: 2026-07
The IPD encompasses highly sensitive and confidential data that was collected through significant investment of our resources, both in terms of time and funding. This data is integral to our ongoing research initiatives, which are at a crucial stage of development. Premature sharing could disrupt our research timelines and strategic plans. Additionally, we have not yet established comprehensive safeguards to ensure that the data will be used appropriately by external researchers. Without proper protocols in place, there is a risk of misuse or misinterpretation of the data, which could lead to inaccurate research outcomes and potential reputational damage to our institution. For these reasons, we have decided not to share the IPD at this time.
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Source: ClinicalTrials.gov.
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