摘要
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 activation in stromal-derived factor 1（SDF-1） mediated migration of human epidermal stem cells and its underlying mechanism.Methods Epidermal stem cells were isolated from preputial skin samples by adhering to collagen type Ⅳ after digesting with Dispase Ⅱ and then cultured in vitro in serum-free defined keratinocyte medium（DK-SFM）.Transwell migration assay was used to observe the migration of human epidermal stem cells that were treated with different PKC inhibitors,including chelerythrine chloride,staurosporine and PS-ζ.Western blot analysis was used to analyze the phosphorylation of PKC-ζ upon SDF-1 stimulation at different time phase.Phalloidin-Rhodamine was used to illustrate morphology of cell cytoskeleton by staining of the F-actin.Laser scanning confocal microscopy was used to observe the polarization of human epidermal stem cells following SDF-1 stimulation.Results Pretreatment of human epidermal stem cells with chelerythrine chloride or PS-ζ decreased the migration of human epidermal stem cells to SDF-1.However,treatment with staurosporine did not affect SDF-1-induced chemotaxis.SDF-1 induced PKC-ζ activation,which was demonstrated by the increased phosphorylation of PKC-ζ with time elapsing.PS-ζ inhibited the polarization of human epidermal stem cells induced by SDF-1.Conclusion SDF-1 promotes the directional migration of human epidermal stem cells via inducing the phosphorylation of signaling protein PKC-ζ and affecting the polarization of F-actin.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2011年第4期335-338,共4页
Journal of Third Military Medical University
基金
国家自然科学基金(30600652)~~
