摘要
目的培养人颊黏膜上皮细胞和分离上皮干细胞,在角膜缘干细胞微环境下诱导颊黏膜上皮干细胞转分化,为前者获取足够的种子细胞。方法应用Epilife培养基培养颊黏膜上皮细胞,使用CFSE标记,观察CFSE标记率及标记细胞与未标记细胞的生物学特性。采用免疫荧光检测第2代细胞P63、CK12、CK13的表达。应用酶差速脱壁法联合差速贴壁法分离培养颊黏膜上皮干细胞,观察体外培养生物学特性。采用免疫荧光检测第2氉代纯化细胞P63、CK12、CK13的表达。结果颊黏膜上皮细胞在Epilife培养基中生长良好。CFSE标记率可达100%,标记细胞与未标记细胞生物学特性相似。经联合分离法获得的第2代细胞免疫荧光P63、K13阳性,K12阴性,而未经联合分离法获得的第2代细胞免疫荧光CK13阳性,P63、CK12阴性。结论人颊黏膜上皮细胞在Epilife培养基中生长良好。CFSE是理想的颊黏膜上皮细胞荧光标记物。酶差速脱壁法联合差速贴壁法纯化了颊黏膜上皮干细胞,为转分化为角膜上皮干细胞获取了足够的种子细胞。
Objective To cultivate human buccal mucosal epithelial cells and i- solate epithelial stem cells, and obtain enough seed ceils to induce buccal mucosal epithelial stem cells transdifferentiating into corneal epithelial stem cells in the microenvironment of corneal limbal stem cells. Methods Buccal mucosal epithelial cells were cultivated with Epilife medium and labeled with CFSE dye. Labeling rate and biological characters of epithelial cells with and without fluorescent labeling were observed. Expression of P63, CK12 and CK13 of passage 2 cells were detected by immunofluorescence. Stem cells were isolated by combination of differential detachment and rapid adhering method and its biological characters were studied. Expression of P63, CK12 and CK13 of purified passage 2 cells were detected by immunofluorescence. Results Buccal mucosal epithelial cells grew well in the Epilife medium. The labeling rate of CFSE reached 100%. Epithelial cells labeled with CFSE showed similar biological characters. Passage 2 cells obtained with combined isolation method were positive with immunofluorescent staining of P63, CK13 and negative of CK12, while passage 2 cells without using that method were positive of CK13 and negative of P53, CK12. Canclusian Human buccal mucosal epithelial cells grow well in the Epilife medium. CFSE dye is an ideal fluorescent marker. The combination of differential detachment and rapid adhering method purify the buccal mucosal epithelial stem cells, which provide enough seed cells to transdifferentiate into corneal epithelial stem cells.
出处
《眼科新进展》
CAS
北大核心
2014年第6期518-522,共5页
Recent Advances in Ophthalmology
基金
广东省眼科学国家重点实验室基金(编号:2012B031-800112)~~
关键词
颊黏膜上皮干细胞
CFSE
差速脱壁法
差速贴壁法
buccal mucosal epithelial stem cell
CFSE
differential detachment method
rapid adhering method
