新生大鼠肺泡Ⅰ型细胞的原代培养及鉴定

Primary culture and identification of type Ⅰ alveolar epithelial cells from neonatal rats

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 develop the isolation and purification method for type Ⅰ alveolar epithelial cells（AEC Ⅰ） of neonatal rats,in order to provide cell culture models for in vitro studies of AECⅠ cells and their roles in lung injury.Methods AECⅠ were collected from Lung lung tissues of 3-day（P3） neonatal rats using elastase and collagenase digestion method and then using monoclonal anti-T1-α antibodies and immunomagnetic beads to select the positive cell for primary cuture.Cell viability was evaluated by trypan blue dye before seeding into culture dishes.Growth status and morphology of attached cells were observed with inverted phase contrast microscope.AECⅠ cells were identified and distinguished from contaminating cells by morphologic criteria and their immunostaining characteristics for T1-α,aquaporin-5 and pro-surfactant protein C（proSP-C）.Results The yield was ~1.4×106 per rat.The purities of AECⅠ was（90.5±3.3）%.The viability of these cell preparations were（93.4±3.0）%.Cells adhered,grew as colonies,and formed a thin monolayer in Day 4-6 of culture.Freshly isolated cell preparations were positive for AQP5 and negative for proSP-C.After 4 days of culture,AECⅠ cells showed positive immunofluorescence for AQP5 primarily.Conclusions The purified AEC Ⅰ with sufficient viability and purity can be used for functional studies.It also offers an important cell culture model to the field of lung injury and repair,and alveolar type Ⅰ cell biology.