差速粘附方法分选表皮干细胞及其评价

Evaluation of the approach to isolate epidermal stem cells by adhering to type Ⅳcollagen

目的 评价细胞外基质差速粘附法在分选表皮干细胞中的作用。方法 人角质形成细胞接种在Ⅳ 型胶原涂被的培养皿上进行粘附试验。粘附细胞为实验组,未粘附细胞及未分选的细胞分别为对照组1和对照组2。三组细胞分别进行克隆形成率测定、细胞周期测定和细胞超微结构观察;应用免疫细胞化学、免疫荧光和流式细胞仪检测β1整合素、细胞角蛋白K19、外皮蛋白(Involucrin)等标记物的表达。结果 与对照组1相比,实验组细胞较小,细胞表面的微绒毛较长而密。实验组细胞克隆形成率明显高于对照组1(P<0.05)。β1整合素、K19、Involucrin在不同组别和不同代次的细胞间表达有显著差别。细胞周期分析显示,实验组细胞86.9％处于G0/G1,8.8％在S期。对照组1和对照组2的细胞处于G0/G1期分别为74％和79.5％,处于S期分别为19.2％和12.3％。结论 细胞外基质差速粘附法可用于分选表皮干细胞。但要达到较高纯度,还需辅以其它手段。

Objective Epidermis is one of the tissues in human body that is maintained through proliferation of stem cells. One approach to isolate epidermal stem cells is established on the basis of their rapid adhesion to extracellular matrix (ECM) proteins. Our study was designed to evaluate this method. Methods Keratinocytes were isolated from human circumcised foreskins of young age. Type Ⅳcollagen coated dishes were used to enrich keratinocyte stem cells. Cells adherent to the coated dishes within 20 min were harvested as experimental group (group A). The nonadherent cells and those of non selected cells were regarded as control group 1 ( group B) and group 2 ( group C). The colony-forming efficiency (CFE) , morphological, immunocytochemical and immunofluorescent and Flow cy-tometer methods were adopted. Results Morphologically, epidermal stem cells are smaller and have fewer organ-elles. The CFE in group A was higher than group B (P <0. 05). The difference in the expression of integrin β1, , K19 and involucrin in groups A, B, C is statistically significant ( P < 0. 01). Quantification of cell cycle profile revealed that about 86.9% of group A cells were in G0/G1 , 8.8% in S, and 4.3% in G2/M. While in the groups B and C, 74% and 79.5% were in G0/G1 , 19.2% and 12.3% in S, 8.2% and 6.8% in G2/M respectively. Conclusion Selective adhesion of keratinocytes to type Ⅳ collagen is a feasible approach to isolate epidermal stem cells.