酶差速脱壁法联合差速贴壁法分离人颊黏膜上皮干细胞

发布时间：2018-04-20 14:27

本文选题：颊黏膜上皮干细胞 + CFSE　；参考：《眼科新进展》2014年06期

【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2l 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。
[Abstract]:Objective to culture and isolate epithelial stem cells from human buccal mucosa, and to induce the transdifferentiation of epithelial stem cells from buccal mucosa under the microenvironment of limbal stem cells, so as to obtain enough seed cells for the former. Methods buccal mucosal epithelial cells were cultured on Epilife medium. The labeling rate of CFSE and the biological characteristics of labeled cells and unlabeled cells were observed by CFSE labeling. The expression of P63, CK12, CK13 was detected by immunofluorescence. The buccal mucosal epithelial stem cells were isolated and cultured by enzyme differential dewalling method and differential adherent method, and the biological characteristics were observed in vitro. The expression of P63, CK12, CK13 was detected by immunofluorescence. Results the labeled rate of buccal mucosal epithelial cells in Epilife medium was 100%, and the biological characteristics of labeled cells were similar to those of unlabeled cells. The second generation of cell immunofluorescence (P63K13) was positive for K12 negative, but the second generation of cell immunofluorescence CK13 positive for P63 / CK12 was negative. Conclusion Human buccal epithelial cells grow well in Epilife medium. CFSE is an ideal fluorescent marker for buccal mucosal epithelial cells. The buccal epithelial stem cells were purified by enzyme differential dewalling method and differential adherent method, and sufficient seed cells were obtained for corneal epithelial stem cells to be transformed into corneal epithelial stem cells.
【作者单位】：南华大学附属第一医院眼科;中山大学中山眼科中心;
【基金】：广东省眼科学国家重点实验室基金(编号:2012B031-800112)~~
【分类号】：R772.2

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