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目的分离人气管上皮细胞,并进行气液界面(air-liquid interface,ALI)培养,为呼吸道病毒研究提供良好的细胞模型。方法采用低温消化法分离人气管上皮细胞,用预包被胶原的0.4μm Transwell培养皿对传代后的气管上皮细胞进行ALI培养。利用倒置显微镜观察细胞生长状态,免疫细胞化学染色和免疫组化鉴定培养细胞的生长和分化情况,同时测定细胞分化过程中的跨上皮电阻(trans epithelial electrical resistance,TEER)。结果分离培养的气管上皮细胞光镜下细胞形态及免疫荧光角蛋白染色阳性,证实培养细胞为气管上皮细胞。ALI培养后的人气管上皮细胞的形态学、MUC5AC蛋白、Ⅳ型β-微管蛋白(β-tubulinⅣ)的表达及紧密连接和假复层的形成情况均与人气管组织结构类似。结论低温消化法可成功分离到活性高的上皮细胞。ALI培养的上皮细胞形态和功能与体内接近,且能较长时间维持其正常形态及功能,为呼吸道相关疾病的研究提供了理想的平台。
Objective To isolate human bronchial epithelial cells and culture them with air-liquid interface (ALI) to provide a good cell model for respiratory virus research. Methods Human bronchial epithelial cells were isolated by cryogenic digestion, and passaged tracheal epithelial cells were cultured in ALI using collagen coated 0.4μm Transwell plates. The growth of cells was observed by inverted microscope, the growth and differentiation of cultured cells were identified by immunocytochemistry and immunohistochemical staining, and the trans-epithelial electrical resistance (TEER) was measured. Results The cultured tracheal epithelial cells were stained by immunofluorescence and immunofluorescence. The cultured cells were tracheal epithelial cells. The morphology of human bronchial epithelial cells cultured in ALI, the expression of MUC5AC protein, the expression of β-tubulin Ⅳ and the formation of tight junctions and pseudostratified tissues were similar to those of human tracheal tissue. Conclusion Low temperature digestion method can successfully isolated high activity epithelial cells. ALI cultured epithelial cells close to the shape and function, and can maintain its normal form and function for a long time, for the study of respiratory diseases provides an ideal platform.