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首页> 外文期刊>Pigment cell research >Quantitative analysis of MC1R gene expression in human skin cell cultures
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Quantitative analysis of MC1R gene expression in human skin cell cultures

机译：人体皮肤细胞培养物中MC1R基因表达的定量分析

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摘要

To address the issue of melanocortin-1 receptor (MC1R) expression in non-melanocytic cells, we have quantitatively evaluated the relative expression levels of both MC1R mRNA and protein in a subset of different cell types. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) at high cycle numbers, we detected MC1R mRNA in all cell types examined, including human embryonic kidney-293 (HEK 293) cells, a cell type widely used as a negative control in melanocortin expression studies. Quantitative real-time PCR revealed the highest levels of MC1R transcripts were in melanocytic cells, whereas the keratinocyte and fibroblast cell cultures examined had only a low level of expression, similar to that of HEK 293 cells. Antibody mediated detection of MC1R protein in membrane extracts demonstrated exogenous receptor in MC1R transfected cell lines, as well as endogenous MC1R in melanoma cells. However, radioligand binding procedures were required to detect MC1R protein of normal human melanocytes and no surface expression of MC1R was detected in any of the non-melanocytic cells examined. This was consistent with their low level of mRNA, and suggests that, if present, the levels of surface receptor are significantly lower than that in melanocytes. The capacity of such limited levels of MC1R protein to influence non-melanocytic skin cell biology would likely be severely compromised. Indeed, the MC1R agonist [NIe(4), D-Phe(7)] alpha-melanocyte stimulating hormone (NDP-MSH) was unable to elevate intracellular cyclic adenosine monophosphate (cAMP) levels in the keratinocyte and fibroblast cells examined, whereas a robust increase was elicited in melanocytes. Although there are a variety of cell types with detectable MC1R mRNA, the expression of physiologically significant levels of the receptor may be more restricted than the current literature indicates, and within epidermal tissue may be limited to the melanocyte.
机译：为了解决非黑素细胞中黑皮质素-1受体（MC1R）表达的问题，我们定量评估了不同细胞类型的子集中MC1R mRNA和蛋白质的相对表达水平。使用高循环数的半定量逆转录聚合酶链反应（RT-PCR），我们在所有检查的细胞类型中检测到MC1R mRNA，包括人类胚胎肾293（HEK 293）细胞，该细胞类型广泛用作阴性细胞黑皮质素表达研究中的对照。实时定量PCR分析显示，MC1R转录本水平最高的是黑素细胞，而所检测的角质形成细胞和成纤维细胞培养物的表达水平却很低，与HEK 293细胞相似。抗体介导的膜提取物中MC1R蛋白的检测证明了MC1R转染细胞系中的外源受体，以及黑素瘤细胞中的内源性MC1R。但是，需要放射性配体结合程序来检测正常人黑素细胞的MC1R蛋白，并且在所检查的任何非黑素细胞中均未检测到MC1R的表面表达。这与其低水平的mRNA一致，并表明，如果存在的话，表面受体的水平明显低于黑素细胞。这种有限水平的MC1R蛋白影响非黑素细胞皮肤细胞生物学的能力可能会受到严重损害。确实，MC1R激动剂[NIe（4），D-Phe（7）]α-黑素细胞刺激激素（NDP-MSH）不能提高所检查的角质形成细胞和成纤维细胞中细胞内环状腺苷单磷酸（cAMP）的水平，而黑素细胞引起强烈的增加。尽管存在多种可检测到MC1R mRNA的细胞类型，但生理上显着水平的受体表达可能比当前文献所显示的受到更多限制，并且表皮组织内的表达可能仅限于黑素细胞。

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