闭塞性细支气管炎中肺祖细胞增殖功能的变化研究
发布时间：2018-08-18 11:45
【摘要】：目的:闭塞性细支气管炎(Obliterative bronchiolitis,OB)是以终末呼吸性细支气管的损伤和不可逆性闭塞为特征的气道炎症性病变,主要临床表现为进行性呼吸困难和咳嗽。OB通常发生在接受移植术后的患者,尤其是实体器官或者骨髓移植,肺功能检查往往提示阻塞性气流受限,临床称之为闭塞性细支气管炎综合征(Bronchiolitis Obliterans Syndrome,BOS)。其组织病理学特征包括,气道上皮细胞和上皮下结构的炎症与损伤,纤维组织过度增殖,气道狭窄阻塞。随着终末期肺病的发病率不断上升,肺移植术后的BOS发生率随之逐年升高,其发病机制的研究已成为目前的热门领域。研究发现BOS患者气道上皮包括基底细胞在内的细胞的损伤,而间质细胞对肺上皮的支持功能对上皮修复至关重要,成纤维细胞正是间质细胞中非常重要的一种。本研究的目的在于探究闭塞性细支气管炎中,肺成纤维细胞对肺祖细胞支持能力的变化。方法:用酶消化的方法获得小鼠肺脏单细胞悬液,流式分选出肺祖细胞(lung progenitor cells,LPCs)。来自正常对照和BOS患者的肺成纤维细胞分别与小鼠肺祖细胞混合培养,观察肺祖细胞的增殖情况,对克隆直径大于100微米的细胞群进行计数。计算两组样本中肺祖细胞的克隆形成效率(colony formation efficiency,CFE),比较两组肺祖细胞的克隆形成能力。数据以平均值±标准差(M±SD)来表示,两组样本之间的比较采用t检验法,P0.05*认为差异有统计学意义。结果:1.小鼠肺祖细胞的分选:使用流式细胞仪分选出肺祖细胞。CD31/34/45-APC-CY7负选择内皮细胞、基质细胞和造血细胞,Ep CAM-PECY7阳性标记上皮细胞,其中上皮细胞中Sca-1-APC和GFP-FITC双阳性标记的为肺祖细胞。2.肺祖细胞的增殖情况:流式分选的肺祖细胞和闭塞性细支气管炎患者来源的肺成纤维细胞共培养,正常人肺成纤维细胞作为对照。培养的第4天和第6天后分别在倒置荧光显微镜下观察肺祖细胞生长。和正常人肺成纤维细胞共培养时,肺祖细胞形成多个克隆,而和闭塞性细支气管炎患者来源的肺成纤维细胞共培养时,细胞克隆明显减少。3.肺祖细胞克隆的计数:统计两组样本中直径大于100微米的克隆数量,比较两组样本肺祖细胞克隆形成率(每100个细胞中克隆形成数量)。相对于基础培养基组,添加SB431542组肺祖细胞克隆形成效率升高。与来自BOS患者的成纤维细胞共培养的肺祖细胞克隆形成效率显著低于正常对照组(P0.05)。结论:本研究建立了小鼠肺祖细胞与BOS患者肺成纤维细胞共培养的模型,研究表明,在闭塞性细支气管炎的发生过程中,肺成纤维细胞对肺祖细胞的支持能力受到损害,气道上皮的修复功能缺陷可能导致持续的气道炎症,最终造成呼吸性终末支气管的纤维性狭窄及不可逆性阻塞。
[Abstract]:Objective: Obliterative bronchiolitis obliterans (OB) is an inflammatory lesion of the airway characterized by terminal respiratory bronchiolitis and irreversibly occlusive bronchiolitis. The main clinical manifestations are progressive dyspnea and cough. OB usually occurs after transplantation, especially in solid organs or bone marrow transplants. Pulmonary function tests often indicate obstructive airflow limitation. Clinically called (Bronchiolitis Obliterans Syndrome bronchiolitis syndrome (BOS). The histopathological features include inflammation and injury of airway epithelial cells and subepithelial structure, hyperproliferation of fibrous tissue and obstruction of airway stenosis. With the increasing incidence of end-stage lung disease, the incidence of BOS after lung transplantation increases year by year, and the study of its pathogenesis has become a hot field. It is found that the airway epithelium including basal cells are damaged in patients with BOS, and the supporting function of interstitial cells to pulmonary epithelium is very important for epithelial repair. Fibroblasts are one of the most important cells in the interstitial cells. The aim of this study was to investigate the changes of lung fibroblast support to lung progenitor cells in bronchiolitis obliterans. Methods: single cell suspension of mouse lung was obtained by enzyme digestion, and lung progenitor cell (lung progenitor cells were selected by flow cytometry. Lung fibroblasts from normal control and BOS patients were co-cultured with mouse lung progenitor cells to observe the proliferation of lung progenitor cells and to count the clones larger than 100 micron in diameter. The clone forming efficiency of lung progenitor cells (colony formation) was calculated in two groups, and the clone forming ability of lung progenitor cells was compared between the two groups. The data were expressed as mean 卤standard deviation (M 卤SD), and the difference between the two groups was statistically significant by t-test method (P0.05 *). The result is 1: 1. Sorting of mouse lung progenitor cells: lung progenitor cells. CD31 / 34 / 45-APC-CY7 negative selective endothelial cells, stromal cells and hematopoietic cells were labeled with EP CAM-PECY7 by flow cytometry. Proliferation of lung progenitor cells: lung progenitor cells and pulmonary fibroblasts derived from patients with bronchiolitis obliterans were co-cultured and normal human lung fibroblasts were used as control. The growth of lung progenitor cells was observed under inverted fluorescence microscope on day 4 and day 6, respectively. When co-cultured with normal human lung fibroblasts, lung progenitor cells formed multiple clones, but when co-cultured with pulmonary fibroblasts derived from patients with bronchiolitis obliterans, the number of cell clones decreased significantly. Count of lung progenitor cell clone: count the number of clones in two groups of samples whose diameter is more than 100 micron, compare the clone forming rate of lung progenitor cell between two groups of samples (number of clone formation per 100 cells). Compared with the basal medium group, the colony forming efficiency of lung progenitor cells was increased in SB431542 group. The colony forming efficiency of lung progenitor cells co-cultured with fibroblasts from BOS patients was significantly lower than that of normal controls (P0.05). Conclusion: the co-culture model of mouse lung progenitor cells and lung fibroblasts in BOS patients was established. The results showed that the ability of lung fibroblasts to support lung progenitor cells was impaired during the development of obliterative bronchiolitis. Defects in the repair of airway epithelium may lead to persistent airway inflammation, resulting in fibrous stenosis and irreversible obstruction of the respiratory terminal bronchus.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.21
[Abstract]:Objective: Obliterative bronchiolitis obliterans (OB) is an inflammatory lesion of the airway characterized by terminal respiratory bronchiolitis and irreversibly occlusive bronchiolitis. The main clinical manifestations are progressive dyspnea and cough. OB usually occurs after transplantation, especially in solid organs or bone marrow transplants. Pulmonary function tests often indicate obstructive airflow limitation. Clinically called (Bronchiolitis Obliterans Syndrome bronchiolitis syndrome (BOS). The histopathological features include inflammation and injury of airway epithelial cells and subepithelial structure, hyperproliferation of fibrous tissue and obstruction of airway stenosis. With the increasing incidence of end-stage lung disease, the incidence of BOS after lung transplantation increases year by year, and the study of its pathogenesis has become a hot field. It is found that the airway epithelium including basal cells are damaged in patients with BOS, and the supporting function of interstitial cells to pulmonary epithelium is very important for epithelial repair. Fibroblasts are one of the most important cells in the interstitial cells. The aim of this study was to investigate the changes of lung fibroblast support to lung progenitor cells in bronchiolitis obliterans. Methods: single cell suspension of mouse lung was obtained by enzyme digestion, and lung progenitor cell (lung progenitor cells were selected by flow cytometry. Lung fibroblasts from normal control and BOS patients were co-cultured with mouse lung progenitor cells to observe the proliferation of lung progenitor cells and to count the clones larger than 100 micron in diameter. The clone forming efficiency of lung progenitor cells (colony formation) was calculated in two groups, and the clone forming ability of lung progenitor cells was compared between the two groups. The data were expressed as mean 卤standard deviation (M 卤SD), and the difference between the two groups was statistically significant by t-test method (P0.05 *). The result is 1: 1. Sorting of mouse lung progenitor cells: lung progenitor cells. CD31 / 34 / 45-APC-CY7 negative selective endothelial cells, stromal cells and hematopoietic cells were labeled with EP CAM-PECY7 by flow cytometry. Proliferation of lung progenitor cells: lung progenitor cells and pulmonary fibroblasts derived from patients with bronchiolitis obliterans were co-cultured and normal human lung fibroblasts were used as control. The growth of lung progenitor cells was observed under inverted fluorescence microscope on day 4 and day 6, respectively. When co-cultured with normal human lung fibroblasts, lung progenitor cells formed multiple clones, but when co-cultured with pulmonary fibroblasts derived from patients with bronchiolitis obliterans, the number of cell clones decreased significantly. Count of lung progenitor cell clone: count the number of clones in two groups of samples whose diameter is more than 100 micron, compare the clone forming rate of lung progenitor cell between two groups of samples (number of clone formation per 100 cells). Compared with the basal medium group, the colony forming efficiency of lung progenitor cells was increased in SB431542 group. The colony forming efficiency of lung progenitor cells co-cultured with fibroblasts from BOS patients was significantly lower than that of normal controls (P0.05). Conclusion: the co-culture model of mouse lung progenitor cells and lung fibroblasts in BOS patients was established. The results showed that the ability of lung fibroblasts to support lung progenitor cells was impaired during the development of obliterative bronchiolitis. Defects in the repair of airway epithelium may lead to persistent airway inflammation, resulting in fibrous stenosis and irreversible obstruction of the respiratory terminal bronchus.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.21
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