全反式维A酸对UVB照射的A375细胞酪氨酸酶代谢及铜锌超氧化物歧化酶的影响

目的 探讨全反式维A酸（ATRA）对中波紫外线（UVB）照射的人A375黑素瘤株细胞黑素含量、酪氨酸酶及铜锌超氧化物歧化酶（Cu/ZnSOD）的影响。方法 将培养的A375细胞分为6组：ATRA + UVB组：A375细胞照射UVB后加入全反式维A酸；氢醌 + UVB组：照射UVB后加入氢醌；UVB组：仅照射UVB；ATRA组：A375细胞加入ATRA；氢醌组：A375细胞加入氢醌；阴性对照组：A375细胞只加入相应溶媒，既不照射UVB，也不加入药物。分别培养，于24 h、48 h和72 h测定黑素含量及酪氨酸酶活性；于培养24 h， Western 印迹检测酪氨酸酶蛋白的变化，实时定量PCR法分别检测酪氨酸酶和Ｃu/Zn SOD在mRNA水平的变化。结果 UVB照射的A375细胞加入全反式维A酸后24 h、48 h、72 h黑素含量及酪氨酸酶活性均下降。UVB照射A375细胞后24 h酪氨酸酶蛋白及mRNA测定值分别为0.72 ± 0.070、1.400 ± 0.135，加入全反式维A酸24 h后酪氨酸酶蛋白及mRNA值分别为0.42 ± 0.056（P < 0.01）、0.810 ± 0.062（P < 0.01）；UVB照射A375细胞后24 h Cu/ZnSOD mRNA水平为0.323 ± 0.066，加入全反式维A酸后Cu/ZnSOD在mRNA水平增高为0.625 ± 0.103（P < 0.01）。结论 全反式维A酸能够通过酪氨酸酶途径抑制UVB辐射导致的A375细胞黑素含量增加，并可提高Cu/ZnSOD水平。

Effects of all-trans retinoic ac id on tyrosinase metabolism and Cu/Zn superoxide dismutase mRNA expression in A375 cells irradiated by ultraviolet B

Objective To evaluate the effects of all-trans retinoic acid （ATRA） on melanin content, activity and protein expression of tyrosinase, mRNA expression of Cu/Zn superoxide dismutase （SOD） in A375 cells irradiated with ultraviolet B （UVB）. Methods Cultured A375 cells were classified into 6 groups: ATRA+UVB group treated with ATRA after UVB irradiation, hydroquinone+UVB group treated with hydroquinone after UVB irradiation, UVB group and ATRA group treated with UVB irradiation and ATRA respectively, negative control group receiving no treatment. Melanin content and tyrosinase activity were determined by NaOH solubilization assay and dopa-oxidation assay respectively at 24, 48 and 72 hours after the addition of ATRA into medium. Western blot was performed to detect the protein expression of tyrosinase, and real-time quantitative PCR to measure the mRNA expressions of tyrosinase and Cu/Zn SOD in A375 cells after 24-hour culture with ATRA. Results The melanin content and tyrosinase activity decreased in UVB-irradiated cells after being treated with ATRA for 24, 48 and 72 hours. The protein （gray scale） and mRNA （2- △△Ct value） expression levels of tyrosinase were 0.72 ± 0.070 and 1.400 ± 0.135 respectively at 24 hours after UVB irradiation, decreased to 0.42 ± 0.056 （P < 0.01） and 0.810 ± 0.062 （P < 0.01） respectively after additional treatment with ATRA. The mRNA expression level of Cu/Zn SOD was 0.323 ± 0.066 in A375 cells at 24 hours after UVB irradiation, and increased to 0.625 ± 0.103 （P < 0.01） after additional treatment with ATRA. Conclusion ATRA can suppress UVB-induced increase in melanin synthesis and elevate Cu/Zn SOD level in A375 cells, likely through tyrosinase pathway.