摘要
目的分离培养和鉴定人表皮基底层干细胞。方法中性蛋白水解酶选择性的消化表皮与真皮之间的细胞连接，采用改良的Ⅳ型胶原铺板选择黏附法分离、培养表皮中的干细胞，观察培养第2、4、6天时a6、β1整合素、K19、K14、p63、Nestin、CD34、PCNA及K10在表皮干细胞中的表达差异。结果改良过的Ⅳ型胶原铺板选择黏附法能够有效地促进表皮干细胞的贴壁和伸展。原代分离的表皮基底层干细胞a6、p1整合素，K19，K14，p63，Nestin，CD34及PCNA表达为强阳性而K10不表达。这些细胞具有干细胞的特点，可以形成克隆。随着培养时间的增加，表皮干细胞形态发生变化，a6、β1整合素、K19、K14，表达逐渐减弱，K10表达逐渐增强。此外，在原代分离的细胞中可见单个核样干细胞表达a6、β1整合素，K19，K14，P63，Nestin，CD34及PCNA，这些细胞形态相似体积较表皮基底层干细胞大，胞核为肾形，染色较深。结论中性蛋白水解酶消化合并改良过的Ⅳ型胶原铺板选择黏附法能够高效地分离表皮中的干细胞。分离的细胞具有干细胞的特点，可以形成集落。此外，原代分离培养的表皮干细胞中仍然可见单个核样干细胞，这些细胞形态相似，类似血液系统来源的单核细胞，胞质内均匀分布着粗大的阳性颗粒。单个核样干细胞可能与皮肤的发牛、发育以及创伤修复有关。
Objective To introduce an efficient method to isolate and identify human epithelial stem cells. Methods Human foreskins were obtained from routine circumcisiom surgeries. Improved collagen Ⅳ -coated adhesion methods was used to isolate and culture the epithelial stem cells after neutron-protease selectively digested the dermo-epidermal junctions. Meanwhile, morphological features and identification of these cells were studied by immunostaining on the day 2, 4 and 6. Results The method introduced to isolate the epithelial keratinocyte stem cells was found to be efficient. These small oval cells metabolized slowly and showed some characteristics of adult stem cells. Immunohistochemical staining revealed that these cells were positive for a6 and β1 integrin, keratin 19 and 14, p63, Nestin, CD34 and PCNA, yet negative for keratin 10 expression. With the prolongation of culture period, the expression of a6 and β1integrin, keratin 19 and 14 was decreased gradually, and K10 content increased relatively. Furthermore, there would be some mono-nuelear cells found in our isolation system which were also positive for a6 and β1integrin, keratin 19 and 14, p63, Nestin, CD34 and PCNA. Conclusion The method of collagen Ⅳ-coated adhesion to isolate epithelial basal stem cells is efficient after neutron-protease selectively digesting the dermo-epidermal junctions. The stem cells isolated have some characteristics of adult stem cells and perhaps the potentiality to differentiate and cross-differentiate. Furthermore, there is also evidence that the hematopoietic stem cells would give rise to a new cutaneous stem cell lineage, involving with the skin＇ s formation and differentiation during embryogenesis and homeostasis and repair in adult.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2007年第2期163-165,共3页
Chinese Journal of Experimental Surgery
基金
国家重点基础研究发展计划资助项目（2005CB522603）
关键词
表皮干细胞
单克隆抗体
免疫组织化学
Epithelial stem cells
Monoclonal antibody
Lmmunohistochemistry
