原代人气道基底细胞的分离与培养

A sophisticate technic to isolate and culture primary human airway basal cells from clinical lung samples

目的:构建成熟的原代人气道基底细胞的分离、培养与传代体系。方法:从废弃的供肺或切除的病肺中分离出气管或较大的支气管;采用蛋白酶、DNA酶消化,搔刮气管或支气管内膜分离基底细胞,接种于Purecol包被的培养瓶,Epi XBase培养基培养、传代;免疫荧光染色法鉴定TRP63+KRT5+基底细胞;比较健康捐赠者和病肺来源的原代气道基底细胞活性。结果:成功分离培养38株人原代人气道基底细胞。标本来源宿主中男性21名、女性17名,年龄中位数59岁(2,77)。其中健康捐献者12例,慢性阻塞性肺疾病(COPD)患者11例,特发性肺纤维化(IPF)患者9例,系统性硬化肺纤维化3例,朗罕氏细胞增生症1例,尘肺病1例,特发性肺高压1例。人气道基底细胞镜下呈多边形,成片连接,形似蜂巢,细胞间连接紧密。成熟细胞形态较均一,细胞核大,胞浆内可见颗粒。免疫荧光染色证实为TRP63+KRT5+基底细胞。38株人气道基底细胞第1代细胞活性为81.25%±0.12%。健康捐献者来源(12例)与病肺来源(26例)的细胞株活性无明显统计学差异。结论:本研究中,分离和培养人气道基底细胞成功率高,细胞纯度高,细胞活性也较高,为人气道基底细胞体外试验提供了技术基础。健康捐献者来源与病肺来源的第1代细胞活性无明显统计学差异。

Objective:To construct the isolation and culture system of primary human airway basal cells.Methods:Human trachea or bronchus were isolated from abandoned donor lung or disease lung removed from the patients undergoing the lung transplant surgery.Trachea or bronchus were digested by proteinase and DNAs for 24 h.Then basal cells were isolated by scraping the endomembrane of trachea or bronchus.The cells were seed in purecol coated flasks and cultured in Epix base medium.TRP63+KRT5+basal cells were identified by immunofluorescence staining.The viability of primary airway basal cells from healthy donor lungs and diseased lungs was measured and compared.Results:Thirty-eight lines of primary human airway basal cells were successfully isolated,cultured and expanded.There were 21 males and 17 females with a median age of 59 years(2,77).Among them,there were 12 healthy donors,[1]patients with chronic obstructive pulmonary disease(COPD),9 patients with idiopathic pulmonary fibrosis(IPF),3 patients with systemic sclerosis pulmonary fibrosis,1 patient with Langham cell hyperplasia,1 patient with pneumoconiosis and 1 patient with idiopathic pulmonary hypertension.Under the microscope,human airway basal cells are polygonal,organized like honeycomb,and cells connected with each other closely.Morphology of mature cells were homogeneous,with large nuclei and granules in cytoplasm.TRP63+KRT5+basal cells were confirmed by immunofluorescence staining.The viability of thirty-eight human airway basal cell lines in Passage 1 was 81.25%±0.12%.There was no significant difference in viability between cell lines isolated from healthy donors(12 cases)and patient with lung diseases(26 cases).Conclusion:In this study,human airway basal cells were successfully isolated and cultured using clinical lung samples.The morphology of the basal cells are homogenous and viability of the cells are satisfied,which is very important for in vitro experiments.There was no significant difference in viability between cell lines from healthy donors and lung disease patients.