中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2015年
10期
692-696
,共5页
黑素细胞%紫外线%Wnt信号通路%细胞增殖%黑素合成
黑素細胞%紫外線%Wnt信號通路%細胞增殖%黑素閤成
흑소세포%자외선%Wnt신호통로%세포증식%흑소합성
Melanocytes%Ultraviolet rays%Wnt signaling pathway%Cell proliferation%Melanin synthesis
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、300 mJ/cm2 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、100 mJ/cm2BB-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.100 mJ/cm2 BB-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 ～ 300 mJ/cm2 BB-UVB照射后,细胞增殖率均逐渐降低,BB-UVB剂量＞100 mJ/cm2时细胞存活率＜50％,同时,10～100 mJ/cm2 BB-UVB照射后酪氨酸酶活性逐渐增加,100 mJ/cm2 BB-UVB组黑素含量明显增加,差异有统计学意义(均P＜ 0.05).30、50、100 mJ/cm2BB-UVB照射后,WIF-1 mRNA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、50 mJ/cm2 BB-UVB组WNT5A mRNA表达量均较对照组降低,100 mJ/cm2 BB-UVB组WNT5A mRNA表达量则明显升高(P＜0.05).100 mJ/cm2 BB-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.
目的 探討寬譜中波紫外線(BB-UVB)照射對黑素細胞增殖率、酪氨痠酶活性、黑素含量的影響.方法 分彆用0、10、20、30、40、50、100、200、300 mJ/cm2 BB-UVB照射原代培養的黑素細胞,採用CCK8法測定黑素細胞增殖率、多巴比色法測定酪氨痠酶活性、NaOH溶解法測定黑素含量.分彆用0、30、50、100 mJ/cm2BB-UVB照射黑素細胞,實時熒光定量PCR檢測非經典Wnt通路相關基因mRNA的錶達.100 mJ/cm2 BB-UVB照射黑素細胞,用Western印跡檢測作用前後非經典Wnt通路相關基因蛋白的錶達.多組間比較採用單因素方差分析,兩組間比較採用獨立樣本t檢驗.結果 與對照組相比,10 ～ 300 mJ/cm2 BB-UVB照射後,細胞增殖率均逐漸降低,BB-UVB劑量＞100 mJ/cm2時細胞存活率＜50％,同時,10～100 mJ/cm2 BB-UVB照射後酪氨痠酶活性逐漸增加,100 mJ/cm2 BB-UVB組黑素含量明顯增加,差異有統計學意義(均P＜ 0.05).30、50、100 mJ/cm2BB-UVB照射後,WIF-1 mRNA的錶達均較對照組逐漸減少,JNK、MITF、RAC1、TYR的錶達均較對照組逐漸升高,而30、50 mJ/cm2 BB-UVB組WNT5A mRNA錶達量均較對照組降低,100 mJ/cm2 BB-UVB組WNT5A mRNA錶達量則明顯升高(P＜0.05).100 mJ/cm2 BB-UVB照射後,WIF-1蛋白的錶達量較對照組降低,WNT5A、JNK、MITF、RAC1、TYR蛋白錶達較對照組升高(P＜0.05).結論 紫外線照射降低黑素細胞增殖率,提高黑素細胞酪氨痠酶活性和黑素含量.WIF-1基因可能抑製黑素生成.WIF-1基因錶達降低可能通過非經典通路Wnt蛋白的綜閤作用激活JNK/MITF/TYR通路,最終促進黑素閤成.
목적 탐토관보중파자외선(BB-UVB)조사대흑소세포증식솔、락안산매활성、흑소함량적영향.방법 분별용0、10、20、30、40、50、100、200、300 mJ/cm2 BB-UVB조사원대배양적흑소세포,채용CCK8법측정흑소세포증식솔、다파비색법측정락안산매활성、NaOH용해법측정흑소함량.분별용0、30、50、100 mJ/cm2BB-UVB조사흑소세포,실시형광정량PCR검측비경전Wnt통로상관기인mRNA적표체.100 mJ/cm2 BB-UVB조사흑소세포,용Western인적검측작용전후비경전Wnt통로상관기인단백적표체.다조간비교채용단인소방차분석,량조간비교채용독립양본t검험.결과 여대조조상비,10 ～ 300 mJ/cm2 BB-UVB조사후,세포증식솔균축점강저,BB-UVB제량＞100 mJ/cm2시세포존활솔＜50％,동시,10～100 mJ/cm2 BB-UVB조사후락안산매활성축점증가,100 mJ/cm2 BB-UVB조흑소함량명현증가,차이유통계학의의(균P＜ 0.05).30、50、100 mJ/cm2BB-UVB조사후,WIF-1 mRNA적표체균교대조조축점감소,JNK、MITF、RAC1、TYR적표체균교대조조축점승고,이30、50 mJ/cm2 BB-UVB조WNT5A mRNA표체량균교대조조강저,100 mJ/cm2 BB-UVB조WNT5A mRNA표체량칙명현승고(P＜0.05).100 mJ/cm2 BB-UVB조사후,WIF-1단백적표체량교대조조강저,WNT5A、JNK、MITF、RAC1、TYR단백표체교대조조승고(P＜0.05).결론 자외선조사강저흑소세포증식솔,제고흑소세포락안산매활성화흑소함량.WIF-1기인가능억제흑소생성.WIF-1기인표체강저가능통과비경전통로Wnt단백적종합작용격활JNK/MITF/TYR통로,최종촉진흑소합성.
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/cm2.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/cm2.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/cm2.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/cm2, 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/cm2.Furthermore, tyrosinase activity gradually increased in melanocytes after irradiation with BB-UVB at 10-100 mJ/cm2 compared with the control group, and the increase was statistically significant at the radiation dose of 100 mJ/cm2 (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) and tyrosinase (TYR) mRNA expression levels increased in melanocytes after irradiation with BB-UVB at 30, 50 and 100 mJ/cm2 (all P ＜ 0.05);the WNT5A mRNA expression significantly decreased in melanocytes irradiated with 30 and 50 mJ/cm2 BB-UVB, but increased in those irradiated with 100 mJ/cm2 BB-UVB (all P ＜ 0.05).The radiation with 100 mJ/cm2 BB-UVB significantly decreased the expression of WIF-1 protein, but enhanced the expressions of WNT5A, JNK, MITF, RAC1 and TYR proteins in melanocytes compared with the control group (all P ＜ 0.05).Conclusions BB-UVB can decelerate the proliferation of, elevate tyrosinase activity and melanin level in, melanocytes.The WIF-1 gene may inhibit melanogenesis, and the decrease in its expression may promote melanogenesis by activating the JNK/MITF/TYR pathway through the combined effects of proteins involved in non-canonical Wnt pathways.
