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

作　　者：李铁征[1] 李泽鸿[1] 李月红[1] 邱业峰[1] 张培因[2] 王树君[3] 朱平[1] 

机构地区：[1]中国人民解放军军需大学研究所,长春130062 [2]吉林大学基础医学部分子生物学教研室,长春130021 [3]长春生物制品研究所,长春130062

出　　处：《中国生物制品学杂志》2005年第3期200-204,共5页Chinese Journal of Biologicals

摘　　要：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.

关 键 词：黑色素瘤 恶性 检测 刺激素 RT-PCR方法 MCIR基因 DNA序列测定 黑素 cDNA 细胞表面受体 扩增片段 细胞膜 R蛋白 分析鉴定 分析结果 特异结合 扩增产物 特异片段 必要条件 配体 分析法 放射 克隆 质粒 测序 表达 

分 类 号：Q78[生物学—分子生物学] TQ658[化学工程—精细化工]