胎鼠肺泡Ⅱ型上皮细胞的分离及原代培养.docVIP

胎鼠肺泡Ⅱ型上皮细胞的分离及原代培养 付红敏，许 峰，黄 波，杨 鸣，符跃强，方 芳，匡凤梧 （重庆医科大学儿童医院PICU，重庆 400014） 摘 要：目的 探讨胎鼠肺泡Ⅱ型上皮细胞（AECⅡ）的分离、纯化及原代培养方法，为有关胎儿肺发育及新生儿肺部疾病的研究奠定基础。方法 采用胰酶结合胶原酶的消化方法，分离肺组织细胞成份，然后经差速离心和差速贴壁的方法纯化AECⅡ，进行原代培养；通过台盼蓝染色检测细胞活力，倒置相差显微镜观察细胞生长特点及形态特征，透射电镜鉴定，改良巴氏染色检测细胞纯度以及免疫荧光技术检测表面蛋白C（SPC）的表达。结果 每3～5只胎鼠可获得AECⅡ(36±5)×106，活力(98±2)％。镜下观察原代AECⅡ呈岛状生长，外观呈多边形或立方形。透射电镜可见特征性的板层小体，改良巴氏染色见胞质内有较多颗粒，纯度为(96±3)％，呈现SPC绿色免疫荧光的细胞占96％以上。结论 利用胰酶和胶原酶消化，以及差速离心和差速贴壁的方法可成功分离出高产量、高纯度的胎鼠AECⅡ，能满足体外进一步实验的需要。 关键词：胎鼠；肺泡Ⅱ型上皮细胞；原代培养；分离 中图分类号：R714.5；R714.7；Q813.1 Isolation and primary culture of typeⅡ alveolar epithelial cells from fetal rat lung FU Hong-min, XU Feng, HUANG Bo, YANG min, FU yueqiang, FANG Fang, KUANG Feng-wu. Pediatrics Intensive Care Unit, Children’s Hospital, Chongqing University of Medical Sciences, Chongqing 400014, China 【Abstract】 Objective To develop the isolation and purification technology for typeⅡ alveolar epithelial cells (AECⅡ) of fetal rat, thus providing experimental means for the 基金项目：国家自然科学基金面上项目(基金号 National Natural Science Foundation(NO. 高等学校博士学科点专项科研基金项目（基金号：20070631010） Research Fund for the Doctoral Program of Higher Education (NO. 20070631010 ) study of lung development or neonatal lung disease. Methods Lung tissues of 19-day fetal rats were digested with trypsin and collagenase, then purified for AECⅡwith different centrifugal force and repeated attachment. Cell viability was evaluated by typran inclusion dying before plating into six-well flask. Growth status and shape of attached cells were observed with inverted phase contrast microscope. AECⅡ were identified by electron microscope and its percentage was assessed by modified Papanicolaou dying and immunofluorescence, the latter aiming to detect expression of surfactant protein C(SPC) in AECⅡ. Results The total amount of cells we harvested from 3 to5 fetal rat was (36±5)×106 with a viability of (98±2)％. Cells were like polygonal and connected like island under microscope．AECⅡ was ascert