Bronchiolitis Obliterans Syndrome (BOS)
Conditions
Keywords
Bronchiolitis Obliterans Syndrome, Airway epithelium, Airway basal cells, Inflammatory response
Brief summary
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.
Detailed 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.
Interventions
PROCEDURETransplantation 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.
PROCEDUREBronchoscopic brushing
The collected samples are digested with tissue collagenase for the culture of airway basal cells (BCs).
GENETICSingle - 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.
GENETICCell 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.
GENETICIdentification 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.
GENETICGene 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.
PROCEDUREPrevention 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.
Sponsors
Haikou Affiliated Hospital of Central South University Xiangya School of Medicine
Study design
Allocation
NON_RANDOMIZED
Intervention model
PARALLEL
Primary purpose
SUPPORTIVE_CARE
Masking
NONE
Intervention model description
In this experiment, bronchoscopic brushing was used to collect epithelial mucosal tissues from the diseased lung area and the relatively healthy lung lobe area of the patients. When conditions permit, alveolar lavage fluid specimens of the patients were collected simultaneously to isolate distal small airway stem cells. Allelic samples from healthy volunteers with no obvious airway abnormalities were used as parallel controls. The collected samples were digested with tissue collagenase and then used for the culture of airway BCS.
Eligibility
Sex/Gender
ALL
Healthy volunteers
Yes
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.
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Ki67 Expression Level in Airway Basal Cells | From enrollment to 32 weeks | 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. |
| Cell Proliferative Activity Assessed by CCK8 Assay | From enrollment to 32 weeks | 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. |
| Expression of Ciliated Cell Marker Ace-Tubulin in Air-Liquid Interface (ALI) Differentiation Culture | From enrollment to 32 weeks | 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. |
| Expression of Goblet Cell Marker MUC5AC in Air-Liquid Interface (ALI) Differentiation Culture | From enrollment to 32 weeks | 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 Ciliated Cell Marker Ace-Tubulin in NSG Mouse Xenograft Model | From enrollment to 32 weeks | 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. |
| Expression of Goblet Cell Marker MUC5AC in NSG Mouse Xenograft Model | From enrollment to 32 weeks | 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. |
| Survival Rate of Ferrets After Airway Basal Cell Transplantation | From enrollment to 32 weeks | 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. |
| Body Weight Changes in Ferrets After Airway Basal Cell Transplantation | From enrollment to 32 weeks | 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. |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| Expression of Airway Basal Cell Markers p63 and Krt5 by Immunofluorescence | From enrollment to 32 weeks | Immunofluorescence staining of transformation protein 63 (p63) and keratin 5 (Krt5), two specific markers for airway basal cells. The percentage of p63-positive and Krt5-positive cells among total cultured cells will be assessed. Positive expression of both markers confirms the identity of cultured cells as airway basal cells, ensuring cell source quality for subsequent experiments. |
Countries
China
Contacts
PRINCIPAL_INVESTIGATORXiaoyang Yang, DM
Department of Hematology, Haikou People's Hospital
Outcome results
None listed