组织块法结合胰蛋白酶消化法培养分离人口腔黏膜上皮细胞和成纤维细胞  

Cultivation and isolation of epithelium and fibroblasts of human oral mucosa with tissue explants method combined with enzymatic digestion method

作　　者：邵晓琳[1] 赵雨[2] 刘慢慢[1] 孟凡皓 刘英[3] 张韬[1] 

机构地区：[1]中国医学科学院北京协和医学院北京协和医院口腔科,北京100730 [2]中国医学科学院北京协和医学院北京协和医院病理科,北京100730 [3]中国医学科学院基础医学研究所生物化学与分子生物学系,北京100005

出　　处：《中国卫生检验杂志》2016年第8期1076-1079,1083,共5页Chinese Journal of Health Laboratory Technology

基　　金：北京市自然科学基金资助项目(7142135);国家科技支撑计划资助项目(2012BAI12B00)

摘　　要：目的介绍组织块法结合胰蛋白酶消化法培养、分离人口腔黏膜上皮细胞和成纤维细胞。方法选择10例患者第三磨牙拔除时切除的龈瓣,大小为(1×0.5)cm^2/块。将带有上皮层和固有层的组织块种植于培养瓶中,待组织块细胞增殖后,通过胰蛋白酶消化法分离获得原代上皮细胞和成纤维细胞。采用免疫荧光和HE染色进行细胞鉴定。结果组织块植入后的第2 d^3 d可看到组织块周边出现新分裂增殖的细胞,14 d细胞增殖达到0.5 cm^1.0 cm后,进行初次传代。胰蛋白酶消化法可初步分离出上皮细胞和成纤维细胞。通过在不同培养基中的传代培养,2种细胞可以获得进一步纯化。结论组织块法结合胰蛋白酶消化法能快速高效的培养分离口腔上皮细胞和成纤维细胞,是一种实用的口腔黏膜细胞培养方法。Objective To cultivate and isolate human oral musosal epithelial cells and fibroblasts by tissue explants method and enzymatic digestion method. Methods The tissues sized（ 1 × 0. 5） cm^2 were collected from retromolar trigone while the impacted tooth being extracted in 10 patients. The tissue was explanted with epithelial layer and lamina propria together. The epithelial cells and fibroblasts were dissociated by trypsin digestion method after tissue cells proliferated. The cells were identified with immunofluorescence technique and Hematoxylin- eosin staining. Results After 2- 3 days the tissue explanted,new cultivated cells were seen around the tissues. 14 d later,subculture could be taken when the radius of cellular proliferation was among 0. 5 cm- 1. 0 cm. The epithelial cells and fibroblasts could be dissociated by trypsin digestion method. The two kinds of cells were purified in different culture medium. Conclusion The tissue explant method combined with enzymatic digestion method is an adoptable way to culture oral mucosal cell,which could cultivate and isolate human oral mucosa epithelium and fibroblasts efficiently and rapidly.

关 键 词：细胞培养 口腔黏膜 上皮细胞 成纤维细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]