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目的研究半硫芥(2-氯乙基乙基硫醚,2-chloroethyl ethyl sulfide,CEES)染毒对角质形成细胞迁移的影响以及miR-34a作用机制。方法体外建立CEES染毒HaCaT细胞模型,采用化学合成miR-34a抑制物转染HaCaT细胞沉默miR-34a,荧光显微镜观察细胞转染效率,细胞划痕实验检测细胞迁移能力,Western印迹检测细胞分化标志物K5和K10、ERK1/2总蛋白及磷酸化水平改变。结果采用0.5 mmol/L CEES染毒后HaCaT细胞迁移显著降低。细胞形态、干性标志物K5和分化标志物K10没有明显变化。miR-34a沉默可以增加细胞迁移能力,迁移相关的信号蛋白ERK1/2通路激活,ERK1/2信号通路抑制剂U0126使用后可以显著降低细胞迁移。结论 miR-34a沉默可以显著增加角质形成细胞迁移,并部分逆转CEES的抑制作用,miR-34a沉默可能部分是通过ERK1/2信号通路激活引起染毒细胞迁移增加。
Objective To investigate the effect of 2-chloroethyl ethyl sulfide (CEES) on the migration of keratinocytes and the mechanism of miR-34a. METHODS: HaCaT cells were established by CEES in vitro. MiR-34a was transfected into HaCaT cells using miR-34a inhibitor. Cell transfection efficiency was observed by fluorescence microscopy. Cell migration was assessed by cell scratch assay. Cell differentiation markers K5 and K10, ERK1 / 2 total protein and phosphorylation levels change. Results The migration of HaCaT cells was significantly reduced with 0.5 mmol / L CEES. Cell morphology, K5, a marker of differentiation, and K10, a marker of differentiation, did not change significantly. miR-34a silencing can increase cell migration, activation of signaling pathway ERK1 / 2 pathway activation, ERK1 / 2 signaling pathway inhibitor U0126 can significantly reduce cell migration. Conclusion Silencing of miR-34a can significantly increase the migration of keratinocytes and partly reverse the inhibitory effect of CEES. The silencing of miR-34a may partly result in the increase of migration of cells exposed through ERK1 / 2 signaling pathway.