摘要：
Objective To evaluate the effect of latanoprost on cell proliferation of and melanogenesis in human epidermal melanocytes,and to explore its mechanism.Methods Latanoprost was added into the 254 medium to prepare latanoprost solutions at different concentrations of 10-5,10-6 and 10-7 mol/L.In vitro cultured human epidermal melanocytes were divided into 4 groups to be cultured with media containing no latanoprost (control group) or 10-5,10-6 and 10-7 mol/L latanoprost for 48 hours.Cell counting kit-8 (CCK8) assay was performed to evaluate the proliferative activity of melanocytes,dopa oxidation assay to estimate the activity of tyrosinase.Sodium hydroxide (NaOH)-lysis method was used to determine the content of melanin,and Masson-Fontana staining to observe the number and distribution of melanin granules.Westernblot analysis and real-time fluorescence-based quantitative PCR were performed to determine the protein and mRNA expression of melanogenesis-related genes including microphthalmia-associated transcription factor (MITF),tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1).Comparison among the 4 groups and multiple comparisons were done by using one-way analysis of variance and least significant difference (LSD)-t test.Results Compared with the control group,the 10-6-,10-5-mol/L latanoprost groups showed significantly increased proliferative activity of melanocytes (1.064 ± 0.172 and 1.078 ± 0.080 vs.0.784 ± 0.015;t =3.289,3.454 respectively,both P ＜ 0.05),increased activity of tyrosinase (0.510 ± 0.017 and 0.454 ± 0.009 vs.0.355 ± 0.041;t =6.139,3.939 respectively,P ＜ 0.01 or 0.05),and increased content of melanin (t =7.232,5.967,both P ＜ 0.01).However,there were no significant differences in the proliferative activity of melanocytes,activity of tyrosinase or content of melanin between the 10-7-mol/L latanoprost group and control group (all P ＞ 0.05).Masson-Fontana staining showed more and darker melanin granules on melanocyte dendrites in the 10-5-,10-6-,10-7-mol/L latanoprost groups than in the control group,and the color of melanin granules changed from light brown to black brown along with the increase in the concentration of latanoprost.The mRNA expression of MITF increased along with the increase in the concentration of latanoprost (P ＜ 0.01),and the protein expression of MITF wassignificantly higher in the 10-6,10-5-mol/L latanoprost groups than in the control group and 10-7-mol/L latanoprost group (all P ＜ 0.01).The 10-6-mol/L latanoprost group showed significantly increased mRNA and protein expression of TYR and TYRP1 compared with the control group,10-7-,10-5-mol/L latanoprost groups (all P ＜ 0.01).Conclusion Latanoprost can increase the proliferation of human epidermal melanocytes,and promote tyrosinase activity and melanogenesis likely by enhancing the mRNA and protein expression of MITF,TYR,TYRP1.%目的 探讨拉坦前列素对人表皮黑素细胞增殖与黑素合成功能的影响及相关机制.方法 将拉坦前列素加入254培养基配制成104、10-6、10-7 mol/L浓度分别作用于体外培养的人表皮黑素细胞48 h,对照组用不加拉坦前列素的培养基处理.采用CCK8法检测黑素细胞增殖,多巴比色法检测酪氨酸酶活性,NaOH溶解法检测黑素合成水平,Masson-Fontana染色法观察细胞黑素颗粒数量及分布,Western印迹及荧光定量PCR分别检测黑素合成相关基因小眼畸形相关转录因子(MITF)、酪氨酸酶(TYR)、酪氨酸酶相关蛋白1(TYRP1)及mRNA的表达.采用单因素方差分析法(ANOVA)和LSD-t检验法分别进行多组间比较及组间两两多重比较.结果 10-6、10-5 mo1/L拉坦前列素组黑素细胞的增殖水平分别为1.064±0.172、1.078±0.080,均显著高于对照组(0.784±0.015,t值分别为3.289、3.454,均P＜0.05);酪氨酸酶活性分别为0.510±0.017、0.454±0.009,亦显著高于对照组(0.355±0.041,t值分别为6.139、3.939,P＜0.01或0.05);黑素含量亦显著增加(LSD-t=7.232、5.967,均P＜ 0.01).而10-7 mol/L拉坦前列素组与对照组相比黑素细胞的增殖水平、酪氨酸酶活性、黑素含量差异均无统计学意义(均P＞ 0.05).Masson-Fontana染色法显示,10-5、10-6、10-7 mol/L拉坦前列素组黑素细胞树突上的黑素颗粒均较对照组数量更多、颜色更深,且随拉坦前列素浓度升高,黑素颗粒的显色从淡棕色加深至黑褐色.MITF mRNA的表达随拉坦前列素浓度的升高而增加(高浓度组与低浓度组比较,均P＜ 0.01),且10-6、10-5 mol/L拉坦前列素组MITF蛋白表达量均显著高于对照组和10-7 mol/L拉坦前列素组(均P＜0.01).10-6 mol/L拉坦前列素组TYR mRNA和蛋白的表达亦均显著高于对照组、10-7、10-5 mol/L拉坦前列素组(均P＜0.01).10-6 mol/L拉坦前列素组TYRP1 mRNA和蛋白的表达均显著高于对照组、10-7、10-5 mol/L拉坦前列素组(均P＜0.01).结论 拉坦前列素可促进人表皮黑素细胞的增殖,并可能通过促进MITF、TYR、TYRP1 mRNA和蛋白的表达促进酪氨酸酶活性和黑素合成.
