多巴胺通过PKR通路诱导黑素细胞PIG1凋亡

Dopamine Induced Apoptosis of PIG1 Melanocyte Cells Involving Activation of PKR Pathway

目的探讨多巴胺诱导黑素细胞PIG1凋亡的机制。方法利用300μmol/L、500μmol/L、1000μmol/L和1500μmol/L多巴胺处理黑素细胞PIG124小时后,以结晶紫法检测细胞活性变化,利用流式细胞术分析AnnexinⅤ/PI双荧光标记细胞的凋亡变化。500μmol/L多巴胺处理黑素细胞1h、3h、6h和12h后收集细胞全蛋白,免疫印迹法检测双链RNA依赖的蛋白激酶PKR、磷酸化双链RNA依赖的蛋白激酶P-PKR和磷酸化真核细胞蛋白质翻译起始因子P-eIF2α水平变化。结果细胞活性随着多巴胺浓度升高而降低,呈剂量依赖关系(P<0.01)。细胞凋亡随多巴胺浓度升高而增加,并呈剂量依赖关系。500μmol/L多巴胺处理后PKR水平未发生明显变化,PKR和eIF2α磷酸化水平明显增加。结论多巴胺诱导的黑素细胞凋亡与PKR通路的激活相关。

Objective To investigate the apoptosis-inducing activity and the molecular mechanisms of dopamine （DA） on human melanocyte cell line PIG1 in vitro. Methods After exposed to different concentration of dopamine for 24h, the viability of melanocyte cell line PIG1 was detected by crystal violet assay. The apoptosis of PIG1 cells was assessed with Annexin V and PI double staining. After treated with 500μmol/L of DA for dif- ferent time, cells were lysed with alysis buffer. PKR, P-PKR, P-eIF2et were detected by Western blot. Resuits Comparing with the experimental control group, DA showed significant inhibiting effect on the viability of PIG1 cells in a dose dependent manner （P 〈0. 01 ）. The apoptosis rate of PIG1 cells was obviously increased. Protein of PKR had no significant change, however, P-PKR and P-eIF2α significantly increased. Conclusion DA induces the apoptosis of PIG1 cells involving activation of PKR pathway.