siRNA抑制A375细胞中黑色素形成

Suppression of Melanin Formation of A375 Cells by Small Interfering RNA

以小干扰RNA(siRNA)技术抑制恶性黑素瘤细胞株A375的黑素皮质素受体1(melanocortin-1-receptor,MC1R)、小眼球相关转录因子(microphthalmia-associated transcription factor,MITF)基因表达,从而抑制细胞黑色素的生成。通过RT-PCR检测MC1R、MITF、酪氨酸酶(tyrosinase,TYR)、酪氨酸酶相关蛋白l(tyrosinase related protein-1,TRP-1)基因mRNA水平的变化,通过测定黑色素含量的变化检测蛋白水平的改变。并用台盼蓝活细胞计数法检测其对细胞增殖的影响。经RT-PCR检测,特异性siRNA作用的靶基因显著下调,黑色素含量明显降低。细胞增殖的变化说明,脂质体包裹的siRNA对A375细胞的毒性远小于熊果苷等化学合成药物。结果显示,利用siRNA对A375细胞中靶基因的调控,有效的抑制了黑色素的生成,siRNA技术与传统的调控黑色素的方法(化学合成物——褪色剂)比较,具有用量小、抑制效率高、细胞毒性低等优点。同时,为进一步研究黑色素形成通路中各基因之间的的关系提供了新思路。

RNA interference technique to the study of melanocortin-l-receptor gene （MC1R） and microphthalmia-associated transcription factor （MITF） expression in human malignant melanoma cell line A375, there by inhibiting the production of melanin. The change in MC 1R, MITF, tyrosinase （TYR） and tyrosinase related protein-1 （TRP-1） mRNA level is detected by RT-PCR. The change of protein level is detected by measuring melanin content. The effect for cell proliferation is detected by using trypanblue exclusion. RT-PCR detects target genes that observably decrease with the effect by specificity small interfering RNA （siRNA） and with the obviously decrease in Melanin content, which informs us that the cytotoxicity of siRNA encapsulated by liposome toward A375 is far less than arbutin and other synthetic drugs. The control in melanoma cells by using siRNA inhibits effectively the production of melanin, Compared with the traditional method （chemical compounds fading agent ） of control melanin, it has the advantage in small amount, high inhibition efficiency, low cytotoxicity, and so forth. Meanwhile, the pathway for further study of melanin formation of the inter-relationship between the key gene has opened up a new path.