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目的探讨信号蛋白PKC-ζ在间质细胞衍生因子1(stromal-derived factor 1,SDF-1)趋化人表皮干细胞定向迁移中的作用及机制。方法Ⅳ型胶原快速分选,无血清培养基(DK-SFM)培养表皮干细胞,免疫组化染色观察表皮干细胞β1整合素(β1 integrin)和角蛋白19(K19)的表达;Transwell实验观察SDF-1对钙和DAG依赖性和非依赖性PKC亚型抑制剂(chelerythrine chloride)、钙和DAG依赖性PKC亚型抑制剂(staurosporine)和PKC-ζ特异性抑制剂(PS-ζ)处理后人表皮干细胞的趋化作用;Western blot法检测SDF-1诱导后不同时段的人表皮干细胞PKC-ζ的磷酸化水平变化;采用Phal-loidin-Rhodamine显示细胞骨架,激光共聚焦显微镜观察PS-ζ对人表皮干细胞极化的影响。结果体外分离培养出人表皮干细胞,免疫组化染色显示,细胞质中β1整合素及K19均呈阳性表达。Transwell实验表明chelerythrine chloride和PS-ζ能够抑制SDF-1趋化的表皮干细胞运动,与阳性对照组比较差异有显著性(P<0.05)。SDF-1作用表皮干细胞能够引起PKC-ζ的磷酸化水平变化,随时间增加逐渐增强;激光共聚焦扫描显示SDF-1处理过的表皮干细胞骨架明显变化,经PS-ζ预处理后细胞骨架变化不明显。结论 SDF-1通过诱导信号蛋白PKC-ζ的磷酸化,影响细胞肌动蛋白的极化,趋化人表皮干细胞的定向迁移。
Objective To investigate the role of PKC-ζ in the directional migration of human epidermal stem cells induced by stromal-derived factor 1 (SDF-1) and its mechanism. Methods The epidermal stem cells were cultured in serum free medium (DK-SFM) and the expressions of β1 integrin and K19 in epidermal stem cells were observed by immunohistochemical staining. The expression of SDF- 1 treatment of post-human calcium and DAG-dependent and independent chelerythrine chloride, calcium and DAG-dependent staurosporine and PKC-ζ specific inhibitor (PS-ζ) The chemotaxis of epidermal stem cells was analyzed by Western blot. The phosphorylation level of PKC-ζ in human epidermal stem cells at different time after SDF-1 induction was detected. Phal-loidin-Rhodamine was used to detect cytoskeleton. PS-ζ was observed with confocal microscopy Effect of human epidermal stem cell polarization. Results Human epidermal stem cells were isolated and cultured in vitro. Immunohistochemical staining showed that β1 integrin and K19 in the cytoplasm were positive. Transwell experiments showed that chelerythrine chloride and PS-ζ can inhibit the epidermal stem cell motility induced by SDF-1 chemotaxis, compared with the positive control group (P <0.05). The effect of SDF-1 on epidermal stem cells induced the change of phosphorylation of PKC-ζ, with the increase of time. The confocal laser scanning confocal microscope showed that the skeleton of SDF-1-treated epidermal stem cells changed obviously. The change of cytoskeleton after PS-ζ pretreatment Not obvious. Conclusion SDF-1 phosphorylates signaling protein PKC-ζ, affecting the polarization of cell actin and chemotaxis to the directional migration of human epidermal stem cells.