Analysis of the Pathogenic Mechanism of Abnormal Airway Basal Cell Function in Bronchiolitis Obliterans Syndrome and Research on Targeted Treatment Strategies
试验速览
- 阶段
- 不适用
- 状态
- 撤回
- 发起方
- 入组人数
- 5
- 试验地点
- 1
- 主要终点
- Ki67 Expression Level in Airway Basal Cells
研究概览
简要总结
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.
详细描述
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.
研究设计
- 研究类型
- Interventional
- 分配方式
- Non Randomized
- 干预模型
- Parallel
- 主要目的
- Supportive Care
- 盲法
- None
入排标准
- 性别
- All
- 接受健康志愿者
- 是
入选标准
- •Patients who have received allogeneic hematopoietic stem cell transplantation and are diagnosed with bronchiolitis obliterans syndrome
排除标准
- •Bronchoscopy consultation is not suitable for patients who are not suitable for bronchoscopy.
研究组 & 干预措施
Gene Editing Group
干预措施: Gene editing (Genetic)
Ferret airway basal cell transplantation group (prevention group)
干预措施: Prevention of ferret airway basal cell transplantation (Procedure)
Ferret airway basal cell transplantation group (treatment group)
干预措施: Transplantation treatment of ferret airway basal cells (Procedure)
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).
干预措施: Bronchoscopic brushing (Procedure)
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.
干预措施: Bronchoscopic brushing (Procedure)
Single - cell clone group
干预措施: Single - cell cloning (Genetic)
Cell Function Identification Group
干预措施: Cell function identification (Genetic)
Local microenvironment change identification group
干预措施: Identification of local microenvironmental changes (Genetic)
结局指标
主要结局
Ki67 Expression Level in Airway Basal Cells
时间窗: 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
时间窗: 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
时间窗: 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
时间窗: 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
时间窗: 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
时间窗: 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
时间窗: 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
时间窗: 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.
次要结局
- Expression of Airway Basal Cell Markers p63 and Krt5 by Immunofluorescence(From enrollment to 32 weeks)