人正常鼻黏膜及鼻息肉黏膜上皮细胞的原代培养及鉴定

Primary Culture and Identification of the Epithelial Cells of Nasal Polyps and Normal Nasal Mucosa

目的探讨人正常鼻黏膜及鼻息肉黏膜上皮细胞的原代培养及鉴定,以备后续对其生物学特性研究之用。方法取2006年-2007年行鼻窦内窥镜下摘除的鼻息肉及行鼻中隔手术切除的正常下鼻甲各5例。标本放入4℃无菌的PBS（配有双抗）溶液中,在超净台中反复冲洗、浸泡,剪成米粒大后转入培养瓶中,用0.1%胶原酶Ⅳ消化过夜。次日取出,轻轻吹打,加入含10%胎牛血清的DMEM/F12（1∶1）培养液,置37℃饱和湿度CO2细胞培养箱中培养。采用ABC贴壁法去除鼻黏膜上皮细胞中的成纤维细胞和红细胞。倒置相差显微镜下观察细胞形态、增殖及生长状况、有无污染。流式细胞仪检测细胞纯度。结果鼻黏膜上皮细胞生长良好,可见4种细胞：纤毛柱状上皮细胞、无纤毛柱状上皮细胞、杯状细胞、基底细胞。透射电镜下鼻息肉黏膜上皮细胞膜表面纤毛较正常鼻黏膜上皮细胞的纤毛数减少,纤毛倒伏,胞质内有空泡形成。经流式细胞仪检测鉴定上皮细胞占90.1%。结论此方法培养的鼻黏膜上皮细胞纯度高,是一种较好的获得原代鼻黏膜上皮细胞的方法。

Objective To investigate the primary culture and identification of epithelial cells from nasal polyps and normal nasal mucosa for further study on their biological features. Methods The specimens of nasal polyps （5 cases） endoscopically resected and surgical resected nosepieces （5 cases） during 2006~2007 were placed into 4℃ sterile phosphate-buffered saline （PBS） solution supplemented with penicillin and streptomycin, washed repeatedly, immersed, cut into pieces, transferred into culture bottles, digested with 0.1% collagenase Ⅳ overnight. On the next day, the samples were removed, pipetted slightly to detach the epithelia, added with 10% DMEM/F12 （1∶1） solution containing fetal bovine serum and cultured in a CO2 incubator with a saturated humidity at 37℃. By using the ABC method, the fibroblasts and erythrocytes were removed. Under an inverted phase microscope, the cells morphology, proliferation, growth and infection were observed. The purity of the cells was analyzed by flow cytometry. Results Nasal mucosa epithelial cells grew well and 4 kinds of cells were found： pillar epithelial cells with cilia, pillar epithelial cells without cilia, goblet cells and basilar cells. Under the transmission electron microscopy, the number of cilia on the surface of mucosal epithelial cell membrane in nasal polyps was less than in that in normal nosepieces. In the nasal mucosa epithelial cells from nasal polyps, the cilia collapsed and there were vacuoles in cytoplasma. Flow cytometry showed the epithelial cells accounted for 90.1%.Conclusion The above mentioned method can be used to obtain primary nasal mucosal epithelial cells with a high purity.