他卡西醇对人表皮黑素细胞增殖、黏附、迁移及c-kit表达的影响

Effect of tacalcitol on the proliferation, adhesion, migration and c-kit mRNA expression of human epidermal melanocytes

目的探讨他卡西醇对体外培养的人表皮黑素细胞增殖、黏附、迁移及c—kitmRNA相对表达的影响。方法用不同浓度的他卡西醇干预体外培养的人表皮黑素细胞，采用四唑氮氢氧化物（XTT）法分别检测培养24、48、72h后黑素细胞的增殖活性；用纤维连接蛋白（FN）包被细胞培养板检测培养72h后黑素细胞的黏附率；用Transwell微孔膜法检测培养24h后黑素细胞在FN上的迁移情况；逆转录-聚合酶链反应（RT—PCR）检测培养72h后黑素细胞c—kitmRNA相对表达量。采用重复测量方差分析及完全随机设计方差分析进行统计学检验。结果重复测量方差分析结果显示，10^-10、10^-9、10^-8、10^-7、10^-6mol／L他卡西醇促进黑素细胞增殖作用的差异有统计学意义（F=9．47，P〈0．01），上述浓度他卡西醇在培养24、48、72h后促进黑素细胞增殖作用的差异有统计学意义（F=14．44，P〈0．01），药物浓度和培养时间之间存在交互作用（F=2．47，P〈0．01），其中10-smol／L他卡西醇与黑素细胞共同培养72h黑素细胞增殖活性最高。10^-8～10^-7mol／L他卡西醇可显著促进黑素细胞在FN上的黏附（均P〈0．01）；10^-9-10^-8mol／L他卡西醇在培养24h能显著促进黑素细胞迁移（均P〈0．01）；RT—PCR显示10^-9～10^-7mol／L他卡西醇在培养72h均能显著增加黑素细胞e—kitmRNA的相对表达量（均P〈0．01）。结论他卡西醇可促进培养的人表皮黑素细胞增殖及在FN上的黏附、迁移作用，并可上调黑素细胞c～kitmRNA的表达。

Objective To evaluate the effect of tacalcitol on the proliferation, adhesion, migration and c-kit mRNA expression of cultured human epidermal melanocytes. Methods Cultured epidermal melanoeytes from the prepuce of adolescent males were treated with various concentrations of tacalcitol. Then, cell proliferation was evaluated by tetrazolium salt （XTI＇） assay after 24, 48 and 72 hours of treatment, adhesive activity by using fibronectin-coated culture plates after 72 hours, migratory activity by Transwell assay using a microporous membrane after 24 hours, and the c-kit mRNA expression was semiquantitatively analyzed by reverse transcription PCR after 72 hours of treatment. Statistical analysis was done by repeated-measure analysis of variance and completely random design analysis of variance. Results As repeated-measure analysis of variance showed, tacaleitol of 10-10, 10-9, 104, 10-7 and 104 mol/L significantly promoted the proliferation of melanocytes （F = 9.47, P 〈 0.01 ）, with significant differences in the promoting effect among various durations of treatment with different concentrations of tacalcitol （F = 14.44, P 〈 0.01 ）, and with significant interaction effect between drug concentration and treatment duration （F = 2.47, P 〈 0.01 ）. The highest proliferation level was observed in melanoeytes treated with tacalcitol of 10-8 mol/L for 72 hours. There was a significant increase in the adhesion rate of human epidermal melanocytes to fibroneetin after treatment with tacalcitol of 104 - 10-7 mal/L for 72 hours （both P 〈 0.01 ）, number of melanocytes migrating through micropore membranes per high-power field （× 200） after treatment with tacalcitol of 10-9 - 104 mol/L for 24 hours （both P 〈 0.01）, and in the c-kit mRNA expression in melanocytes treated with taealcitol of 10-9 - 10-7 mol/L for 72 hours （all P 〈 0.01 ）. Conclusion Tacalcitol can promote melanocytes to proliferate, migrate, express c-kit mRNA, and adhere to fibronectin.