α和β-熊果苷对共培养小鼠黑素细胞黑素生成的影响研究

Effects of α-arbutin and β-arbuutin on melanogenesis in murine melanocytes cocultured with murine keratinocytes

目的比较观察熊果苷分子苯环侧链上的糖苷基团α-和β-构象对共培养小鼠黑素细胞酪氨酸酶活性和黑素含量的影响。方法体外melan-a小鼠黑素细胞（MC）与SP-1小鼠角质形成细胞（KC）共培养体系，两种细胞的初始接种比例为1：5。共培养细胞分别用含有0．04、0．2、1mg／ml的α-熊果苷、β-熊果苷、烟酰胺及10、50、100μmol／L的甲氧沙林（8-MOP）新鲜培养基换液，药物处理共4d。用相差倒置显微镜观察细胞表型的改变；用四甲基偶氮唑盐比色（MTT）法测定细胞增殖率；用放射性核素L-[^14C]-酪氨酸底物标记法测定酪氨酸羟化酶与多巴氧化酶活性；用分光光度计法测定细胞总黑素含量。结果除8-MOP能明显促进共培养细胞（主要是MC）发生增殖外，其他3种化合物对细胞增殖率均无影响。两种熊果苷均能剂量依赖地抑制酪氨酸酶活性和黑素含量。1mg／mlα-熊果苷对酪氨酸酶活性的抑制作用比同等浓度的β-熊果苷强（酪氨酸酶活性：37％±4％vs54％±9％，P=0．034）。经两种熊果苷（1mg／m1）处理的MC树状突延长纤细，黑素产生较对照组明显减少，以α-熊果苷更为明显。相反，100μmol／L8-MOP处理组的MC数目较对照组显著增加，树状突增加，胞质与树状突内有大量黑褐色黑素堆积。结论α-熊果苷能明显抑制共培养小鼠黑素细胞的黑素生成，其临床应用价值有待体内实验进一步确认。

Objective To investigate the effects of α-arbutin and β-arbutin on tyrosinase activity and melanin content in murine melanocytes （MCs） cocultured with murine keratinoeytes （KCs） and to define whether the difference in configuration of glucosidic linkage （s） of hydroquinone glucosides is correlated with their inhibitory activities against tyrosinase. Methods Mouse MCs of the line melan-a and mouse KCs of the line SP-1 were cocultured at a ratio of 1 ： 5. The ceils were treated for 4 days with the compound [ α-arbutin or β-arbutin 0.04, 0.2, or 1 mg/ml, nicotinamide 0.04, 0.2, or 1 mg/ml, and 8- methoxypsoralen （8-MOP） 10, 50, 100 μmol/L respectively]. Phenotypic alterations of MCs cocultured with KCs were examined and photographed by phase-contrast microscopy. The viability/proliferation of the treated ceils was measured by methylthiazol tetrazolium （MTr） assay. The tyrosinase （monophenolase and o-diphenolase） activities were determined by incorporation of radioactive L- [ 14 C ] tyresine substrate into the melanin, and the melanin content was determined by spoctrophotometric assay. Results 1 mg/ml nicotinamide slightly increased the melanin in dendrites and cytoplasm of the MCs. The dendrites of the MCs treated by 1 mg/ml α-arbutin and β-arbutin were long and thin and melanin was less compared with the control group. 8-MOP significantly promoted the proliferation of the melan-a and SP-1 ceils, especially of the melan-a ceils. Nicotinamide, α-arbutin, and β-arbutin did not significantly influence the proliferation of the MCs and KCs. The tyrosinase activity and melanin content were increased by 8-MOP dose-dependently and dramatically inhibited by α-arbutin, and β-arbutin dose-dependently. The inhibitory effect of a-arbutin on tyrosinase was stronger than that of β-arbutin at a dose of 1 mg/ml （the tyrosinase activity：37% ＋4% vs 54% ＋ 9%, P = 0. 034 ）. Conclusion α-arbutin is an effective and safe ingredient for skin-whitening purpose in vitro experiment. Clinical implication remains to be further evaluated by in vivo studies.