宽谱中波紫外线对人表皮黑素细胞非经典Wnt通路的作用研究  

Effects of broadband ultraviolet B on non-canonical Wnt pathways in human epidermal melanocytes

作　　者：王辉[1] 林彤[1] 王千秋[1] 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所,南京210042

出　　处：《中华皮肤科杂志》2015年第10期692-696,共5页Chinese Journal of Dermatology

基　　金：江苏省自然科学基金（BK2012507）

摘　　要：目的探讨宽谱中波紫外线（BB—UVB）照射对黑素细胞增殖率、酪氨酸酶活性、黑素含量的影响。方法分别用0、10、20、30、40、50、100、200、300mJ／cm^BB—UVB照射原代培养的黑素细胞，采用CCK8法测定黑素细胞增殖率、多巴比色法测定酪氨酸酶活性、NaOH溶解法测定黑素含量。分别用0、30、50、100mJ／cm^2BB—UVB照射黑素细胞，实时荧光定量PCR检测非经典Wnt通路相关基因mRNA的表达。100mJ／cm^2BB—UVB照射黑素细胞，用Western印迹检测作用前后非经典Wnt通路相关基因蛋白的表达。多组间比较采用单因素方差分析，两组间比较采用独立样本t检验。结果与对照组相比，10～300mJ／cm^2BB—UVB照射后，细胞增殖率均逐渐降低，BB—UVB剂量〉100mJ／cm^2时细胞存活率〈50％，同时，10～100mJ／cm^2BB—UVB照射后酪氨酸酶活性逐渐增加，100mJ／cm^2BB—UVB组黑素含量明显增加，差异有统计学意义（均P〈0．05）。30、50、100mJ／cm^2BB—UVB照射后，WIF-1mRNA的表达均较对照组逐渐减少，JNK、MITF、RAC1、TYR的表达均较对照组逐渐升高，而30、50mJ／cm。BB—UVB组WNT5AmRNA表达量均较对照组降低，100mJ／cm^2BB—UVB组WNT5AmRNA表达量则明显升高（P〈0．05）。100mJ／cm^2BB—UVB照射后，WIF-1蛋白的表达量较对照组降低，WNT5A、JNK、MITF、RAC1、TYR蛋白表达较对照组升高（P〈0．05）。结论紫外线照射降低黑素细胞增殖率，提高黑素细胞酪氨酸酶活性和黑素含量。WIF-1基因可能抑制黑素生成。WIF-1基因表达降低可能通过非经典通路Wnt蛋白的综合作用激活JNK／MITF／TYR通路，最终促进黑素合成。Objective To investigate the effects of broadband ultraviolet B （BB-UVB） on the proliferation of, tyrosinase activity and melanogenesis in melanocytes. Methods Melanocytes isolated from human foreskin were subjected to primary culture. Some cultured primary melanocytes were irradiated with BB-UVB at 10, 20, 30, 40, 50, 100, 200 and 300 mJ/cm^2. Then, CCK-8 assay was performed to evaluate the proliferative activity of melanocytes, dopa oxidation assay to estimate the activity of tyrosinase, and sodium hydroxide （NaOH）-lysis method was used to determine melanin content. Real-time fluorescence-based quantitative PCR was conducted to measure the mRNA expressions of genes involved in non-canonical Wnt pathways in melanocytes after irradiation with BB-UVB at 30, 50 and 100 mJ/cm^2. Western blot was carried out to determine the expressions of proteins involved in non-canonical Wnt pathways in melanocytes before and after irradiation with BB-UVB of 100 mJ/cm^2. The melanocytes receiving no treatment served as the control group. Statistical analysis was carried out by one-way analysis of variance followed by least significant difference （LSD）-t test for multiple group comparisons and by the independent sample t test for two-group comparisons. Results After irradiation with BB-UVB at 10 - 300 mJ/cm^2, the proliferative activity of melanocytes was gradually reduced compared with the control group （all P 〈 0.05 ）, and the survival rate of melanocytes was less than 50% when the irradiation dose of BB-UVB was higher than 100 mJ/cm^2. Furthermore, tyrosinase activity gradually increased in melanoeytes after irradiation with BB-UVB at 10 - 100 mJ/cm^2 compared with the control group, and the increase was statistically significant at the radiation dose of 100 rnJ/cm^2 （P 〈 0.05）. Compared with the control group, the WIF-1 mRNA expression level decreased, while c-Jun N-terminal kinase （JNK）, microphthalmia-associated transcription factor （MITF）, Ras-related C3 botulinum toxin substrate 1 （RAC 1 �

关 键 词：黑素细胞 紫外线 Wnt信号通路 细胞增殖 黑素合成 

分 类 号：R751[医药卫生—皮肤病学与性病学]