多巴色素异构酶突变对体外培养黑素小体成熟和抗氧化应激能力的影响

Effects of mutation in dopachrome tautomerase on melanosome maturation and anti-oxidative potential in cultured melanocytes

目的研究多巴色素异构酶（Dct）突变对黑素细胞黑素小体成熟和活性氧基（ROS）清除能力的影响。方法用透射电子显微镜技术和Fontana—Masson嗜银染色观察小鼠Dct突变slaty黑素细胞和野生型melan—a黑素细胞胞内黑素小体发育及嗜银着色的优黑素颗粒分布；用分光光度计法和蛋白印迹技术测定酪氨酸酶活性和酪氨酸酶，酪氨酸酶相关蛋白1（Tyrp-1）和Dct蛋白表达水平；用二氯荧光素标记法测定两种黑素细胞在3J／cm^2长波紫外线（UVA）照射前后胞内ROS水平的变化。结果透射电子显微镜观察发现slaty黑素细胞胞内缺乏成熟的Ⅳ期黑素小体，Fontana—Masson嗜银染色也证实slaty黑素细胞胞质几乎完全缺乏嗜银染阳性的优黑素颗粒。与melan—a黑素细胞相比，slaty黑素细胞的离心团块颜色变浅，但slaty细胞的酪氨酸酶活性及蛋白质表达水平与melan—a细胞接近。UVA照射前slaty和melan-a细胞胞内的ROS相对荧光强度分别为8．9±0．7和8．9±2．5，但照射后slaty细胞胞内的ROS相对荧光强度急剧增加，与melan—a黑素细胞相比，差异有统计学意义（18．0±0．3比13．6±3．0，P=0．024）。结论Dct突变导致黑素细胞低色素化表型，影响黑素小体的发育成熟和降低细胞对氧化应激的抵抗能力，尤其是在UVA诱导氧化应激存在时更为明显。

Objective To investigate whether the mutation in dopachrome tautomerase （Dct） affects melanosome maturation and anti-oxidative potential in cultured melanocytes （MCs）. Methods Slaty and melan-a MCs were derived from the skins of neonatal Dct^Sit and C57 BL/6J mice respectively. Their detailed melanosome structures were examined with a transmission electron microscopy （TEM） and their eumelanin granules characterized by Fontana-Masson staining. Furthermore, the tyrosinase activity and three melanogenic proteins, i.e. , tyrosinase, tyrosinase-related protein 1 and Dct, were also measured with a spectrophotometery method or Western blot assay. The level of intracellular reactive oxygen species （ROS） was monitored by 2, 7-dichlorofluorescin diacetate （DCF-DA） labeling. Results Mature stage Ⅳ melanosomes markedly decreased in slaty MCs under TEM. The brownish granules stained with Fontana- Masson silver method were far less in slaty MCs than in melan-a MCs. The cell pellet of slaty MCs was white in color, but the similarities between slaty and melan-a were found in tyrosinase activity and its protein expression. The relative intensity of DCF fluorescence was 8.9 ±0. 7 for slaty melanocytes versus 8.9 ±2. 5 for melan-a melanocytes prior to UVA irradiation, but an abrupt ROS production was merely observed in slaty MCs （18.0 ±0. 3） other than in melan-a MCs （13.6 ± 0. 3 ） after UVA exposure. There was statistical difference between these two cell lines in ROS level upon UVA irradiation （ P = 0. 024）. Conclusion The mutation in Dct causes hypo-pigmented phenotype in cultured slaty MCs, inhibits melanosome maturation and decreases anti-oxidative capacity especially in the presence of UVA-induced oxidative stress.