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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 To evaluate the role of extracellular matrix differential adhesion in the sorting of epidermal stem cells. Methods Human keratinocytes were seeded on collagen type IV coated dishes for adhesion assays. The adherent cells were experimental group, non-adherent cells and unsorted cells were control group 1 and control group 2, respectively. Three groups of cells were measured for clonality, cell cycle and cell ultrastructure. Immunocytochemistry, immunofluorescence and flow cytometry were used to detect β1 integrin, cytokeratin K19, Involucrin and other markers expression. Results Compared with the control group 1, the experimental group cells smaller, longer and dense microvilli on the cell surface. The colony formation rate of experimental group was significantly higher than that of control group 1 (P <0.05). β1 integrin, K19, Involucrin expression in different groups and different generations of cells were significantly different. Cell cycle analysis showed that 86.9% of the cells in the experimental group were in G0 / G1, 8.8% in S phase. Cells in control group 1 and control group 2 were 74% and 79.5% in G0 / G1 phase, respectively, and were 19.2% and 12.3% in S phase respectively. Conclusion Extracellular matrix differential adhesion method can be used to sort epidermal stem cells. However, to achieve higher purity, supplemented by other means.