閉塞性細支氣管炎中肺祖細胞增殖功能的變化研究
發(fā)布時間：2018-08-18 11:45
【摘要】：目的:閉塞性細支氣管炎(Obliterative bronchiolitis,OB)是以終末呼吸性細支氣管的損傷和不可逆性閉塞為特征的氣道炎癥性病變,主要臨床表現(xiàn)為進行性呼吸困難和咳嗽。OB通常發(fā)生在接受移植術(shù)后的患者,尤其是實體器官或者骨髓移植,肺功能檢查往往提示阻塞性氣流受限,臨床稱之為閉塞性細支氣管炎綜合征(Bronchiolitis Obliterans Syndrome,BOS)。其組織病理學(xué)特征包括,氣道上皮細胞和上皮下結(jié)構(gòu)的炎癥與損傷,纖維組織過度增殖,氣道狹窄阻塞。隨著終末期肺病的發(fā)病率不斷上升,肺移植術(shù)后的BOS發(fā)生率隨之逐年升高,其發(fā)病機制的研究已成為目前的熱門領(lǐng)域。研究發(fā)現(xiàn)BOS患者氣道上皮包括基底細胞在內(nèi)的細胞的損傷,而間質(zhì)細胞對肺上皮的支持功能對上皮修復(fù)至關(guān)重要,成纖維細胞正是間質(zhì)細胞中非常重要的一種。本研究的目的在于探究閉塞性細支氣管炎中,肺成纖維細胞對肺祖細胞支持能力的變化。方法:用酶消化的方法獲得小鼠肺臟單細胞懸液,流式分選出肺祖細胞(lung progenitor cells,LPCs)。來自正常對照和BOS患者的肺成纖維細胞分別與小鼠肺祖細胞混合培養(yǎng),觀察肺祖細胞的增殖情況,對克隆直徑大于100微米的細胞群進行計數(shù)。計算兩組樣本中肺祖細胞的克隆形成效率(colony formation efficiency,CFE),比較兩組肺祖細胞的克隆形成能力。數(shù)據(jù)以平均值±標準差(M±SD)來表示,兩組樣本之間的比較采用t檢驗法,P0.05*認為差異有統(tǒng)計學(xué)意義。結(jié)果:1.小鼠肺祖細胞的分選:使用流式細胞儀分選出肺祖細胞。CD31/34/45-APC-CY7負選擇內(nèi)皮細胞、基質(zhì)細胞和造血細胞,Ep CAM-PECY7陽性標記上皮細胞,其中上皮細胞中Sca-1-APC和GFP-FITC雙陽性標記的為肺祖細胞。2.肺祖細胞的增殖情況:流式分選的肺祖細胞和閉塞性細支氣管炎患者來源的肺成纖維細胞共培養(yǎng),正常人肺成纖維細胞作為對照。培養(yǎng)的第4天和第6天后分別在倒置熒光顯微鏡下觀察肺祖細胞生長。和正常人肺成纖維細胞共培養(yǎng)時,肺祖細胞形成多個克隆,而和閉塞性細支氣管炎患者來源的肺成纖維細胞共培養(yǎng)時,細胞克隆明顯減少。3.肺祖細胞克隆的計數(shù):統(tǒng)計兩組樣本中直徑大于100微米的克隆數(shù)量,比較兩組樣本肺祖細胞克隆形成率(每100個細胞中克隆形成數(shù)量)。相對于基礎(chǔ)培養(yǎng)基組,添加SB431542組肺祖細胞克隆形成效率升高。與來自BOS患者的成纖維細胞共培養(yǎng)的肺祖細胞克隆形成效率顯著低于正常對照組(P0.05)。結(jié)論:本研究建立了小鼠肺祖細胞與BOS患者肺成纖維細胞共培養(yǎng)的模型,研究表明,在閉塞性細支氣管炎的發(fā)生過程中,肺成纖維細胞對肺祖細胞的支持能力受到損害,氣道上皮的修復(fù)功能缺陷可能導(dǎo)致持續(xù)的氣道炎癥,最終造成呼吸性終末支氣管的纖維性狹窄及不可逆性阻塞。
[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 鹵standard deviation (M 鹵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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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 鹵standard deviation (M 鹵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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R562.21
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