Studies About Impaired Capacity Of Lung Progenitor Cells In Obliterative Bronchiolitis

Objective: Obliterative bronchiolitis(OB)is an inflammatory airway disease characterized by injury and irreversible obstruction of the terminal respiratory bronchial.The main clinical manifestations are progressive dyspnea and cough.OB usually occurs in patients who have undergone transplantation,especially in solid-organ or bone marrow transplantation.Pulmonary function tests often indicate obstructive airflow limitation,and clinically known as Bronchiolitis Obliterans Syndrome(BOS).Its histopathological features include inflammation and injury of airway epithelial cells and subepithelial structures,excessive proliferation of fibrous tissue,and bronchial obstruction.Recent years,with the increasing incidence of end-stage lung disease,BOS after lung transplantation raised,its pathogenesis has become the current hot area.Studies have found damage to airway epithelia cells,including basal cells,in BOS patients,however the lung epithelial-supportive capacity of stromal cells of which fibroblasts are a very important kind is essential for pulmonary epithelial repair.This study aimed to investigate the changes of progenitor cell-supportive capacity of lung fibroblasts during the development of OB.Method: The mouse lung single cell suspension was obtained by enzymatic digestion,and lung progenitor cells(LPCs)were sorted by flow cytometry.Lung fibroblasts from normal controls and BOS patients were mixed with mouse lung progenitor cells respectively to observe the proliferation of lung progenitor cells.The colony with diameter greater than 100 microns was counted and compared the colony formation ability of lung progenitor cells through the colony formation efficiency(CFE)of two groups.The data were present as mean ± standard deviation(M ± SD).The Student's t-test was used to test for statistical significance.P values below 0.05 * were considered statistically significant.Results: 1.The isolation of mouse lung progenitor cells: Lung progenitor cells were sorted using flow cytometry.We used CD31 / 34/45-APC-CY7 for negative selection of endothelial cells,stromal cells and hematopoietic cells,then Ep CAM-PE-CY7 positive labeled epithelial cells were selected,lung progenitor cells were enriched by Sca-1-APC and GFP-FITC double staining.2.Proliferation of lung progenitor cells: Mouse lung progenitor cells and human lung fibroblasts from patients with BOS or normal control respectively were co-cultured.The proliferation of lung progenitor cells were observed under inverted fluorescence microscopy on day 4 and day 6.When co-cultured with normal human lung fibroblasts,the lung progenitor cells formed multiple clones,however the clones were significantly reduced when co-cultured with lung fibroblasts from patients with BOS.3.Colony formation efficiency of lung cell clones: Count the number of clones with diameter greater than 100 microns in both groups,and compare the colony formation efficiency of lung progenitor cells(number of clones per 100 cells).Compared with the basal medium group,the clonal formation efficiency of lung progenitor cells in SB431542 group was increased.The colony formation efficiency of lung progenitor cells co-cultured with fibroblasts from patients with BOS was significantly lower than that with the normal control group(P <0.05).Conclusion: In this study,we established a model of co-culture of mouse lung progenitor cells and lung fibroblasts from BOS patients.We conclude that the progenitor cell-supportive capacity of fibroblasts is impaired in the development of OB.The dysfunction of airway epithelium repair caused persistent airway inflammation that eventually lead to fibrous stenosis and irreversible obstruction of the respiratory terminal bronchus.