人恶性黑色素瘤A375细胞黑素刺激素受体(MC1R)的检测

Detection of Alpha Melanocyte Stimulating Hormone Receptor of Human Melanoma Cell Line A375

目的检测人恶性黑色素瘤A375细胞膜上MC1R蛋白及其cDNA,为研究黑色素瘤细胞表面受体及靶向配体奠定基础。方法利用放射配体分析法检测人恶性黑色素瘤A375细胞膜上的MC1R蛋白;用RTPCR方法扩增人恶性黑色素瘤A375细胞MC1R的cDNA,并克隆至pMD18T质粒,进行酶切分析鉴定及扩增片段DNA序列测定。结果放射配体分析结果表明,125IαMSH可与人恶性黑色素瘤A375细胞膜特异结合。RTPCR结果显示,扩增产物的大小与MC1R的cDNA相符。测序结果证实扩增片段为MC1R基因的特异片段。结论证实人恶性黑色素瘤A375细胞膜上表达MC1R蛋白。MC1R基因的成功克隆为其进一步研究提供了必要条件。

Objective To detect the alpha melanocyte stimulating hormone receptor MC1R of human melanoma cell line A375 and lay a foundation of further study on surface receptor and target ligand of melanoma cells.Methods Detect the MC1R protein on the membrane of human melanoma A375 cells by radioligand bindng assay using ~ ~125 I-labeled α-melanocyte stimulating hormone(MSH).Amplify the cDNA of A375 cell MC1R by RT-PCR,insert into plasmid pMD-18T.Digest the recombinant plasmid by restriction endonuclease and sequence the amplified fragment.Results Radioligand binding assay showed that ~ ~125 I-labeled α-MSH was specifically bound to the membrane of A375 cells.The amplified fragment showed a length consistent with that of MC1R cDNA and a sequence identical to that of MC1R gene reported in GenBank.Conclusion MC1R protein was successfully expressed on the membrane of human melanoma cell line A375.It provided a basis for the further study on MC1R.