摘要
目的:探讨酶消化分离法原代培养人鼻黏膜上皮细胞的实验步骤和技术要点,以提高原代培养成功率,为后续实验提供良好的培养模型。方法:在酶消化分离细胞、无血清培养法的基础上,对取材处理、消化酶应用等步骤进行改进,如翻揭分离获取黏膜层、消化中加入I型DNA酶(DNase type I)、胶原酶消化后含黏膜块的溶液中直接加入胰蛋白酶,以及应用不包被的培养皿培养等。原代培养的鼻黏膜上皮细胞经免疫荧光染色细胞角蛋白(CK)8/18进行鉴定。结果:鼻黏膜原代培养细胞生长良好,6-8d汇合可用于后续实验。CK8/18免疫荧光染色阳性鉴定为上皮源性细胞。结论:酶消化分离细胞、无血清培养法可成功建立人鼻黏膜上皮细胞原代培养模型,对取材处理、酶消化等步骤进行上述改进,可提高原代培养成功率,并获得一定数量级,以及纯度较高和可靠的原代上皮细胞。
Objective:To highlight the key points of primary culture of human nasal epithelial cells by enzymatieal dissociation for high achievement ratio, and to establish a successful primary culture model for subsequent experiments. Method:Primary culture of human nasal epithelial cells was performed with enzymatical dissociation of isolated tissue in serum-free medium. On the basis of this method, some improvements were subjected, such as stripping mucosal epithelium from adjacent connective tissue, applying DNase type I to digesting procedure, adding trypsin directly to the eollagenase solution containing digested mucosa pieces, employing uncoated culture dishes and so on. Immunofluoreseence with a monoclona[ anti-cytokeratin antibody 8/18 was used to confirm the epithelial nature of the cultured cells. Result: Nasal epithelial cells grew well and confluenced on the 6th to 8th day. Positive expression of eytokeratin(CK)8/18 showed the epithelial property of cultured ceils. Conclusion: Primary culture model of human nasal epithelial cells can be successfully established by enzymatical dissociation. Improvements on processes of material using and enzyme digestion can gain a high achievement ratio and harvest a high purity and certain amount of reliable primary epithelial cells.
出处
《临床耳鼻咽喉头颈外科杂志》
CAS
CSCD
北大核心
2009年第23期1066-1068,共3页
Journal of Clinical Otorhinolaryngology Head And Neck Surgery
基金
国家自然科学基金(No:30772412)
关键词
鼻黏膜
上皮
细胞培养
nasal mucosa
epithelium
cell culture