人原代支气管上皮细胞的简易分离和培养
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摘要
目的建立一种实验环境更贴合人体、方便快捷、细胞活性和纯度较高的支气管上皮细胞原代培养方法。方法通过纤维支气管镜刷检获取新乡市第一人民医院收治的63例患者正常的支气管上皮细胞,采用无血清的支气管上皮细胞培养基(BEGM)进行纯化、分离,行原代培养并传代,锥虫蓝染色,光学显微镜下观察细胞存活状态并测定细胞存活率,通过倒置生物式显微镜和免疫组织化学染色观察鉴定细胞。结果人支气管上皮细胞的平均存活率为(96.8±0.5)%,细胞免疫荧光角蛋白染色阳性,兔抗人细胞角蛋白8单克隆抗体免疫组织化学染色显示细胞质着色为绿色,细胞核复染为蓝色,鉴定为人原代支气管上皮细胞。结论经纤维支气管镜刷检获取并采用无血清BEGM培养人原代支气管上皮细胞操作简便、快捷有效,细胞存活率较高,能够提高上皮细胞纯度,可以为研究呼吸系统疾病提供良好的模型。
Objective To establish a method of primary culture of bronchial epithelial cells with more suitable for human body,convenient,high cell activity and purity. Methods Sixty-three cases were examined by bronchoscopy in the bronchoscopy room of the First People's Hospital of Xinxiang City,and the relative normal bronchial epithelial cells were obtained by brush biopsy. The bronchial epithelial cells were purified and separated by serum-free medium,and then cultured and passaged. Trypan blue staining was carried out. The survival state of the cells was observed under light microscope,and the survival rate was measured. The cells were identified by inverted phase contrast microscope and immunohistochemical staining. Results The survival rate of human bronchial epithelial cells obtained and isolated by fiberoptic bronchoscopy was( 96. 8 ±0. 5) %. The cell morphology and immunofluorescent keratin staining were positive under the inverted biological microscope.Immunohistochemical staining of rabbit anti-human cytokeratin 8 monoclonal antibody showed that the cytoplasm was green and the nucleus was blue,and the cells were identified as primary bronchial epithelial cells. Conclusion A method of obtaining human primary bronchial epithelial cells by bronchoscopic brush biopsy and culturing them with serum-free BEGM is established. This method is simple,rapid and effective,and has high cell survival rate. It can improve the purity of epithelial cells and provide a good model for the study of respiratory diseases.
Objective To establish a method of primary culture of bronchial epithelial cells with more suitable for human body,convenient,high cell activity and purity. Methods Sixty-three cases were examined by bronchoscopy in the bronchoscopy room of the First People's Hospital of Xinxiang City,and the relative normal bronchial epithelial cells were obtained by brush biopsy. The bronchial epithelial cells were purified and separated by serum-free medium,and then cultured and passaged. Trypan blue staining was carried out. The survival state of the cells was observed under light microscope,and the survival rate was measured. The cells were identified by inverted phase contrast microscope and immunohistochemical staining. Results The survival rate of human bronchial epithelial cells obtained and isolated by fiberoptic bronchoscopy was( 96. 8 ±0. 5) %. The cell morphology and immunofluorescent keratin staining were positive under the inverted biological microscope.Immunohistochemical staining of rabbit anti-human cytokeratin 8 monoclonal antibody showed that the cytoplasm was green and the nucleus was blue,and the cells were identified as primary bronchial epithelial cells. Conclusion A method of obtaining human primary bronchial epithelial cells by bronchoscopic brush biopsy and culturing them with serum-free BEGM is established. This method is simple,rapid and effective,and has high cell survival rate. It can improve the purity of epithelial cells and provide a good model for the study of respiratory diseases.
引文
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[13]YUAN C,HE Q,LI J M,et al.Evaluation of embryonic age and the effects of different proteases on the isolation and primary culture of chicken intestinal epithelial cells in vitro[J].Anim Sci J,2015,86(6):588-594.
[14]COMER D M,ELBORN J S.Comparison of nasal and bronchial epithelial cells obtained from patients with COPD[J].PLo S One,2012,7(3):e32924.
[15]高元妹,张平,夏旸,等.人支气管上皮细胞原代培养方法的对比研究[J].实用医学杂志,2011,27(15):2698-2700.
[2]MIKAMI Y,MATSUZAKI H,HORIE M,et al.Lymphotoxinβreceptor signaling induces IL-8 production in human bronchial epithelial cells[J].PLo S One,2014,9(12):e114791.
[3]BORGIE M,LEDOUX F,VERDIN A,et al.Genotoxic and epigenotoxic effects of fine particulate matter from rural and urban sites in Lebanon on human bronchial epithelial cells[J].Environ Res,2015,136:352-362.
[4]TSAO S M,YIN M C.Antioxidative and antiinflammatory activities of asiatic acid,glycyrrhizic acid,and oleanolic acid in human bronchial epithelial cells[J].J Agric Food Chem,2015,63(12):3196-3204.
[5]ALEM F,YAO K,LANE D,et al.Host response during Yersinia pestis infection of human bronchial epithelial cells involves negative regulation of autophagy and suggests a modulation of survival-related and cellular growth pathways[J].Fron Microbiol,2015,6:50.
[6]KIDO H,YOKOGOSHI Y,SAKAI K,et al.Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells:a possible activator of the viral fusion glycoprotein[J].J Biol Chem,1992,267(19):13573-13579.
[7]高元妹,徐军.组织贴块法人的支气管上皮细胞的原代培养[J].国际呼吸杂志,2008,28(7):397-399.
[8]秦晓群,孙秀泓,张长青.联合应用酶消化和机械刷洗提取气道上皮细胞的实验技术[J].湖南医科大学学报,1999,24(1):74-76.
[9]张景熙,李强,白冲,等.经纤维支气管镜刷检人支气管上皮细胞的原代培养[J].第二军医大学学报,2004,25(7):791-792.
[10]WIDDICOMBE J H,COLEMAN D L,FINKBEINER W E.Primary cultures of the dog's tracheal epithelium:fine structure,fluid,and electrolyte transport[J].Cell Tissue Res,1987,247(1):95-103.
[11]REUS A A,MAAS W J,JANSEN H T,et al.Feasibility of a 3D human airway epithelial model to study respiratory absorption[J].Toxicol In Vitro,2014,28(2):258-264.
[12]李茂中,庞立丽,王宏,等.人气管上皮细胞的原代分离及气液界面培养[J].中国生物制品学杂志,2016,29(1):65-69.
[13]YUAN C,HE Q,LI J M,et al.Evaluation of embryonic age and the effects of different proteases on the isolation and primary culture of chicken intestinal epithelial cells in vitro[J].Anim Sci J,2015,86(6):588-594.
[14]COMER D M,ELBORN J S.Comparison of nasal and bronchial epithelial cells obtained from patients with COPD[J].PLo S One,2012,7(3):e32924.
[15]高元妹,张平,夏旸,等.人支气管上皮细胞原代培养方法的对比研究[J].实用医学杂志,2011,27(15):2698-2700.