Objective To investigate DJ-1 expression and protective effect against H2O2-induced oxidative stress in primary human melanocytes.Methods The expression and location of DJ-1 in primary melanocytes were identified by immunofluorescence.After cultured melanocytes were exposed to H2O2 for 24 hours,modified MTT assay was used to assess the proliferation of cells and to choose the suitable concentration of H2O2 for the following experiment.Western blot was used to detect D J-1 expression in melanocytes after being treated with 0.5 mmol/L H2O2 for 24 hours.Some melanocytes were divided into 3 groups to be reversely transfected with PBS(mock control group),non-targeting siRNA(negative control group)and DJ-1 targeting siRNA(DJ-1 group).Optical microscopy was utilized to observe the morphologic changes of transfected melanocytes.At 48 hours after the transfection,the melanocytes were stimulated with H2O2 for 24 hours.Subsequently,modified MTT assay,2′,7′-dichlorofluorescein diacetate(DCFH-DA)and annexin Ⅴ-fluorescein isothiocyanate/propidium iodide were used to determine cell viability,intracellular reactive oxygen species (ROS)level and apoptosis rate respectively.Results DJ-1 was expressed in both melanocyte nucleus and cytoplasm,and predominantly in the nucleus.H2O2 inhibited the cell viability in a dose dependent manner.After treatment with H2O2 of 0.5 mmol/L for 24 hours,the cell viability began to decrease in melanocytes with the expression level of DJ-1 being 2.23 times that in the untreated melanocytes(both P ＜ 0.05).Compared with the mock control group,the dendrites of melanocytes in DJ-1 group were obviously shortened with cytoplasm vacuolization.After 24-hour treatment with H2O2 of 0.5 mmol/L,the cell viability in the DJ-1 group dropped to 35％ of that in the mock control group(P ＜ 0.05),while the intracellular ROS fluorescence intensity( FI) and apoptosis rate were higher in the DJ-1 group than in the mock control group (902 ± 40 vs.529± 32,58q％ ± 6.1％ vs.30% ± 3.8％,both P ＜ 0.05).Conclusion DJ-1 can protect melanocytes against H2O2induced oxidative stress likely by decreasing intracellular ROS production and inhibiting ceU apoptosis.%目的 探讨DJ-1蛋白在原代培养的人黑素细胞中的表达以及抵抗H2O2诱导的氧化应激作用.方法 免疫荧光方法鉴定DJ-1在原代培养黑素细胞中的表达；使用不同浓度H2O2处理黑素细胞24h,改良MTT法选取适宜H2O2浓度为后续实验条件；Western印迹检测H2O2处理组细胞内DJ-1表达变化；采用反向转染方法,实验分为空白对照组(不含siRNA片段)、阴性对照组(转染非特异性siRNA)和实验组(转染DJ-1特异性siRNA).光学显微镜观察细胞贴壁后形态学差异；改良MTT法、荧光探针2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)、膜联蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶(AnnexinⅤ-FITC/PI)分别检测H2O2处理后细胞活力变化、细胞内活性氧(ROS)水平及凋亡比例.结果 免疫荧光显示DJ-1在黑素细胞细胞核和胞质中均表达,以细胞核表达为主；细胞活力随H2O2浓度增高呈剂量依赖关系下降,与对照组比较,0.5 mmol/L H2O2处理24h后,细胞活力开始下降(P＜0.05).做诱导氧化应激的实验条件；Western印迹结果显示,0.5 mmol/L H2O2处理细胞24h后,DJ-1表达增高为H2O2未处理组的2.23倍(P＜0.05).干涉DJ-1表达后,实验组黑素细胞形态与空白对照组相比,树突减少缩短,胞质空泡化改变.使用0.5 mmol/L H2O2处理24h后,siRNA-DJ-1组细胞活力为对照组的35％(P＜0.05).细胞内ROS的荧光密度值(FI)值(902±40)和凋亡比例(58％±6.1％)增高,与空白对照组细胞内ROS的FI值(529±32)和凋亡比例(30％±3.8％)相比,P值均＜0.05.结论 DJ-1在黑素细胞中能够通过降低细胞内ROS 水平和抑制细胞凋亡,从而抵抗H2O2所诱导的氧化应激.
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