培养基诱导B16细胞黑色素合成及机理研究

The Mechanism of Medium Stimulating the Melanin Synthesis in B16 Murine Melanoma Cells

研究不同培养基对B16细胞体外培养生物学特性的影响及其黑色素表达能力与相关分子作用机制.方法:利用不同培养基对B16细胞进行体外连续传代培养,考查培养基对细胞形态、细胞成活率、细胞周期和黑色素合成等方面的影响.同时,借助RT-PCR、Western Blot和双向电泳等技术,研究与黑色素生物合成相关蛋白质的表达水平.结果:B16细胞在Gibco-RPMI-1640-31800和Gibco-DMEM-12800这两种培养基中培养时,细胞外观形态良好、细胞活力强、传代周期短并且均可稳定连续传代培养,两者的细胞周期也一致;B16细胞在GibcoRPMI-1640-31800培养基中基本不合成黑色素,而在Gibco-DMEM-12800培养基能大量合成黑色素.分析B16细胞中三种黑色素合成相关蛋白质(Tyrosinase,Tyrosinase-related protein 1和Tyrosinase-related protein 2)在以上两种培养基中表达的差异,发现这三种蛋白质的m RNA转录水平基本没有差异,在Gibco-DMEM-12800培养基中这三种蛋白质的翻译水平均明显提高,总蛋白质组也更加多样.结论:Gibco-DMEM-12800培养基通过提高Tyrosinase、TRP1和TRP2等三种蛋白质的翻译水平使B16细胞黑色素大量合成.

To study the effect of different media on the biological characteristics of B16 murine melanoma cells in vitro, and the expression mechanism of melanin, B16 cells were subcultured continuously in different media, and their biological characteristics including morphology, cell proliferation, cell cycle were observed. Meanwhile, the transcriptional and translational levels of melanin were investigated by Western Blot, RT-PCR, and two-dimensional electmphoresis. The results indicated that B16 murine melanoma cells had good cellular morphology, high cell viability, rapid proliferation, and could be subcultured continuously in two media, Gibco-RPMI-1640-31800 （RPMI-1640） and Gibco-DMEM-12800 （DMEM）. However, melanin was abundant in the 1~16 cells cultured in DMEM medium, while only small amount of that was produced in RPMI-1640 medium. Notably, there is no difference in the transcriptional levels of the mRNA of tymsinase, Tyrosinase- related protein 1, and Tyrosinase-related protein 2 between two media detected by RT-PCR. But the Western Blot experiment showed that the translational level of these three proteases were obvious higher in DMEM medium than in RPMI- 1640 medium. Additionally, the cells cultured in DMEM medium had more proteomic diversity detected through two- dimensional electrophoresis. These results revealed that B16 cells produced substantial amount of melanin through increasing the translational level of tyrosinase, TRP1 and TRIY2 in B16 murine melanoma ceils in DMEM medium.