圣草酚对角质形成细胞和黑色素瘤细胞共培养体系中黑色素合成的促进作用

Promotional Effect of Eriodictyol on Melanin Synthesis in Co-Culture System of HaCaT Cells and Melanoma Cells

为了研究圣草酚对角质形成细胞(HaCaT)与黑色素瘤细胞(B16)共培养体系中细胞增殖、酪氨酸酶活性、黑色素含量以及酪氨酸酶(TYR)、酪氨酸相关蛋白1(TRP-1)、酪氨酸相关蛋白2(TRP-2)mRNA表达的影响,建立了HaCaT细胞与B16细胞体外共培养体系模拟“表皮黑色素形成单位”,分别采用MTT法测定细胞增殖率、多巴氧化速率法测定细胞酪氨酸酶激活率、氢氧化钠裂解法测定细胞黑色素合成率、实时荧光定量PCR法测定TYR、TRP-1、TRP-2的基因相对表达量。结果表明,圣草酚在低浓度下对共培养体系中的细胞无毒性作用,而且有明显的促增殖作用;12.5~50μmol·L^(-1)圣草酚对共培养体系中细胞酪氨酸酶活性有激活作用(P<0.001),并呈剂量依赖性;12.5~50μmol·L^(-1)圣草酚能显著提高共培养体系中细胞黑色素合成率(P<0.01或P<0.0001);12.5~50μmol·L^(-1)圣草酚能显著提高共培养体系中TYR的基因相对表达量(P<0.01或P<0.001),25~50μmol·L^(-1)圣草酚能显著提高共培养体系中TRP-1、TRP-2的基因相对表达量(P<0.01)。圣草酚可能通过促进TYR、TRP-1、TRP-2的mRNA表达来促进共培养体系中细胞酪氨酸酶活性提升和黑色素合成。

To investigate the effects of eriodictyol on cell proliferation,tyrosinase activity,melanin content,and mRNA expression of tyrosinase(TYR),tyrosine-related protein 1(TRP-1),and tyrosine-related protein 2(TRP-2),we established an in vitro co-culture system of keratinocytes cells(HaCaT)and melanoma cells(B16)to simulate the"epidermal melanogenic units".Moreover,we measured the cell proliferation rate by MTT method,the tyrosinase activation rate by the levodopa oxidation rate method,the melanin synthesis rate by the sodium hydroxide cracking method,and the relative gene expression levels of TYR,TRP-1,and TRP-2 by the real time fluorescence quantitative PCR.The results show that eriodictyol with low concentrations has a non-toxic effect on cells in the co-culture system and has a significant proliferative effect;moreover,12.5-50μmol·L^(-1) eriodictyol can activate the tyrosinase activity in the co-culture system(P<0.001)in a dose-dependent manner;12.5-50μmol·L^(-1) eriodictyol can significantly increase the melanin synthesis rate of cells in the co-culture system(P<0.01 or P<0.0001);12.5-50μmol·L^(-1) eriodictyol can significantly increase the relative gene expression level of TYR in the co-culture system(P<0.01 or P<0.001),25-50μmol·L^(-1) eriodictyol can significantly increase the relative gene expression levels of TRP-1 and TRP-2 in the co-culture system(P<0.01).Eriodictyol may promote the tyrosinase activity and melanin synthesis in the co-culture system by increasing the mRNA expression of TYR,TRP-1,and TRP-2.