胎鼠肺泡Ⅱ型上皮细胞的分离及原代培养

Isolation and primary culture of typeⅡ alveolar epithelial cells from fetal rat lung

目的:探讨胎鼠肺泡Ⅱ型上皮细胞(Alveolar epithelial cell typeⅡ,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(Surfactant proteinc,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。

Objective： To develop the isolation and purification technology for type Ⅱ alveolar epithelial cells（ AEC Ⅱ ）of fetal rat, in order to provide experimental means for the 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 AEC Ⅱ with different centrifugal force and repeated attachment. Cell viability was evaluated by typran inclusion dying before plated into six-well flask. Growth status and shape of attached cells were observed with inverted phase contrast microscope. AEC Ⅱ were identified by electron microscopy and its percentage was assessed by modified Papanicolaou dying and immunofluorescence , the latter aiming to detect expression of surfactant protein C （SPC）in AEC Ⅱ. Results： The total amount of cells we harvested from 3 to5 fetal rat was （36 ± 5 ） × 10^6 with a viability of 98% ± 2%. Cells were like polygonal and connected like island under microscope. AEC Ⅱ was ascertained by lamellar bodies found in cytoplasma under electron microscope and its percentage was 96% ± 3 % identified by modified Papanicolaou dying,the same as the result by immunofluorescence for SPC. Conclusion： Highly Purified and viable AEC Ⅱ could be achieved by our methods and the cell model could be used in further study.