Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors
Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors
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DOI：
10.1073/pnas.93.24.13715
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发表时间：
1996-11-26
影响因子：
11.1
通讯作者：
Hruby, VJ
中科院分区：
文献类型：
--
作者：
Sharma, SD;Jiang, JW;Hruby, VJ
我们开发了两种固相试剂系统的显微镜可视化和表征黑色素细胞刺激激素（MSH）受体的黑色素瘤细胞。通过硫醚或二硫键将多个拷贝的[Nle-4，D-Phe-7]-a-MSH（a-MSH的有效类似物）缀合至微球（乳胶珠）或大球（聚酰胺珠）。通过扫描电子显微镜和光学显微镜检查珠与小鼠和人黑素瘤细胞之间的结合。促黑激素缀合物的结合不限于细胞周期的任何一个阶段。通过几项研究证明了结合的特异性。阴性对照包括非黑素细胞来源的细胞类型（例如，乳腺癌细胞）和缺乏促黑素配体或具有其它连接配体的珠粒。具有二硫键连接的促黑素类似物的珠粒用作直接对照。在珠与黑素瘤细胞孵育期间或之前用DTT处理这些珠（导致MSH类似物从珠中释放）消除了珠与黑素瘤细胞的结合。黑素瘤细胞和促黑素蛋白结合的珠之间的结合相互作用也可以通过预先与未缀合的MSH类似物孵育而消除。在这些实验中，还观察到某些膜受体-激素相关的现象，例如受体-配体复合物的加帽（聚集）。这些结果提供了MSH受体是黑素瘤细胞共有的性质的视觉证据。正常人表皮黑素细胞和角质形成细胞也显示表达促黑素素受体，这与为黑素瘤细胞建立的标准相同。
We developed two solid-phase reagent systems for microscopic visualization and characterization of melanocyte-stimulating hormone (MSH) receptors of melanoma cells. Multiple copies of [Nle-4,D-Phe-7]-alpha-MSH, a potent analog of alpha-MSH, were conjugated to microspheres (latex beads) or macrospheres (polyamide beads) through a thioether or disulfide bond, Binding between the beads and mouse and human melanoma cells was examined by scanning electron microscopy and by light microscopy, Each mouse and human melanoma cell (of all cell lines) evinced binding to the beads. Binding of the melanotropin conjugates was not restricted to any one phase of the cell cycle. Specificity of binding was demonstrated by several studies, Negative controls included cell types of nonmelanocyte origin (e.g., mammary cancer cells) and beads that lacked the melanotropic ligand or had other attached ligands, Beads with a disulfide-linked melanotropin analog served as a direct control. Treatment of these beads with DTT during or before incubation of the beads with melanoma cells (resulting in release of the MSH analog from the beads) eliminated binding of the beads to melanoma cells, Binding interactions between melanoma cells and melanotropin-bound beads also could be abolished by prior incubation with unconjugated MSH analog. During these experiments, certain membrane receptor-hormone associated phenomena, such as capping (aggregation) of the receptor-ligand complex, also were observed, These results provide visual evidence that MSH receptors are a property common to melanoma cells, Normal human epidermal melanocytes and keratinocytes were also shown to express melanotropin receptors by the same criteria established for melanoma cells.