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目的探讨新生大鼠肺泡Ⅰ型上皮细胞(AECⅠ)的分离、纯化及原代培养方法,为主要以AECⅠ损伤为特征的肺部疾病研究提供模型。方法采用弹性蛋白酶联合胶原酶Ⅰ的消化方法,分离肺组织细胞,然后用肺Ⅰ型上皮细胞顶膜蛋白α(T1-α)单克隆抗体及免疫磁珠进行阳性选择的方法纯化AECⅠ,最后进行原代培养;通过锥虫蓝染色检测细胞活力,倒置显微镜观察细胞生长及形态特征,细胞免疫荧光法检测细胞表面特异性抗原的表达。结果每只鼠可获得AECⅠ(1.4±0.4)×106个,纯度90.5%±3.4%,活力93.4%±3.0%。接种于玻璃培养皿48h后倒置显微镜下观察细胞开始粘附伸展,呈克隆样生长,在4～6天时汇合成薄的单层状。免疫荧光显微镜观察新鲜分离的AECⅠ特异标志物水通道蛋白-5(AQP5)阳性,呈现绿色荧光;AECⅡ标志物前表面活性蛋白C(proSP-C)阴性;培养后的AECⅠ免疫荧光染色呈AQP5阳性。结论利用弹性蛋白酶联合胶原酶Ⅰ消化,免疫磁珠进行阳性选择的方法可成功分离出高产量、高纯度的鼠AECⅠ,能满足体外进一步对AECⅠ生理功能的研究,也可为研究AECⅠ损伤修复机制提供细胞培养的模型。
Objective To investigate the isolation, purification and primary culture of alveolar type Ⅰ epithelial cells (AEC Ⅰ) in neonatal rats and to provide a model for the study of lung diseases characterized mainly by AEC Ⅰ injury. Methods Elastase and Collagenase Ⅰ digestion were used to isolate lung cells and then purified with AEC Ⅰ by positive selection with monoclonal antibody against porcine pulmonary epithelial type Ⅰ (T1-α) and immunomagnetic beads. Finally, Primary culture, cell viability was detected by trypan blue staining, cell growth and morphological features were observed by inverted microscope, and cell surface specific antigen was detected by immunofluorescence. Results Each mouse obtained AECⅠ (1.4 ± 0.4) × 106, purity of 90.5% ± 3.4% and activity of 93.4% ± 3.0%. Inoculated in glass dishes 48h after inverted microscope under the microscope the cells began to adhere to stretch, was clonal growth in 4 ~ 6 days confluence into a thin monolayer. Immunofluorescence microscopy showed freshly isolated AEC I-specific aquaporin-5 (AQP5) was positive and showed green fluorescence; AECⅡmarker proSP-C was negative; AECP immunofluorescence staining was AQP5 positive . Conclusion The method of elastase combined with digestion of collagenase Ⅰ and immunomagnetic beads can successfully isolate rat AEC Ⅰ with high yield and purity and can meet the requirement of further studying the physiological functions of AEC Ⅰ in vitro. Provide a model of cell culture.