摘要目的 探讨核因子E2p45相关因子2(Nrf2)核转位对永生化黑素细胞株B10BR细胞生物学活性的影响.方法 构建电转染野生型和nls缺失型nrf2质粒载体至B10BR细胞,结合叔丁基氢醌(TBHQ)和(或)H2O2处理,Western印迹检测Nrf2,his标签蛋白表达,MTr法测定细胞活性,多巴氧化法检测酪氨酸酶活性,Transwell法测定转染细胞的迁移.结果 TBHQ处理的转染组细胞核中Nrf2表达均显著高于TBHQ未处理组(P＜0.01).转染质粒组间及其与未处理组间细胞酪氨酸酶活性差异均无统计学意义(P＞0.05),H2O2处理使2个质粒转染组酪氨酸酶活性较单独质粒转染组显著下降(pcDNA-nrf2,P＜0.05; pcDNA-nrf2△nls,P＜ 0.05).相对于TBHQ未添加H2O2处理的nrf2组,TBHQ显著增强H2O2处理的野生型nrf2质粒转染细胞酪氨酸酶活性(P＜0.05)；相对于TBHQ未添加H2O2处理的nls组,nls缺失型nrf2质粒转染组细胞酪氨酸酶活性差异无统计学意义(P＞0.05).相对于nrf2未转染组,H2O2对野生型nrf2质粒转染组细胞活性的影响无统计学意义(P＞0.05)；相对于两组的未处理组,H2O2引起未转染组及nls缺失型质粒转染组细胞活性显著下降(未处理组,P＜ 0.05；pcDNA-nrf2△nls,P＜0.01).TBHQ可以保护野生型nf2质粒转染细胞免受H2O2引起的氧化损伤,对nls缺失型质粒转染组细胞无明显保护作用.各处理组黑素细胞迁移差异无统计学意义(P＞0.05).结论 Nrf2核转位异常影响黑素细胞的抗氧化能力、黑素细胞活性及酪氨酸酶活性.TBHQ可以激活野生型Nrf2在细胞核中表达水平,增强酪氨酸酶和黑素细胞活性,进而提高细胞的抗氧化水平.

abstractsObjective To investigate the effect of nuclear translocation of E2p45 related factor 2 (Nrf2)on the biological activity of melanocytes.Methods Plasmid vectors containing wild-type nrf2 gene (pcDNA-nrf2) and nls-deleted nrf2 gene (pcDNA-nrf2△nls) were constructed.B10BR normal murine melanocytes were classified into three groups,i.e.,untransfected group,wild-type nrf2 group transfected with pcDNA-nrf2,and mutated nrf2 group transfected with pcDNA-nrf2△nls.Each of the above groups were further divided into three subgroups:control subgroup receiving no treatment,hydrogen peroxide (H2O2) subgroup treated with H2O2 of 200 μmol/L for 24 hours,and combined subgroup pretreated with tert-butyl hydroquinone (TBHQ) followed by treatment with H2O2 of 200 μmol/L for 24 hours.Subsequently,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of cells,dopa oxidation assay to determine tyrosinase activity,Transwell assay to estimate cell migration ability,Western blot to quantify the expressions of Nrf2 and his tag fusion protein.Results TBHQ significantly enhanced the nuclear expression of Nrf2 in B10BR cells transfected with pcDNA-nrf2 or pcDNA-nrf2△nls (both P ＜ 0.01).No significant difference was observed in tyrosinase activity between untreated wild-type nrf2 group,mutated nrf2 group,and untransfected group (P ＞ 0.05).There was a statistical decrease in tyrosinase activity in the two H2O2-treated transfected groups compared with the untreated transfected groups (both P ＜ 0.05),and the decrease was reversed by TBHQ pretreatment in the wildtype nrf2 group (P ＜ 0.05),but not in the mutated nrf2 group (P ＞ 0.05).Further more,the proliferative activity of B10BR cells experienced no obvious changes in the wild-type nrf2 group (P ＞ 0.05),but was significantly reduced in the untransfected group (P ＜ 0.05) and mutated nrf2 group (P ＜ 0.01) after the H2O2 treatment compared with the corresponding untreated groups.TBHQ could protect the pcDNA-nrf2-transfected B10BR cells,but not pcDNA-nrf2△nls-transfected B10BR cells,from H2O2-induced oxidative damage.Transwell assay showed no significant difference in migration ability among these nine groups (P ＞ 0.05).Conclusions Abnormal nuclear translocation of Nrf2 could affect antioxidant activity of,proliferative activity of and tyrosinase activity in melanocytes.TBHQ may enhance the tyrosinase activity in,proliferative activity and antioxidant activity of melanocytes via activating the nuclear expression of wild type Nrf2.