Butts Cherie L, Shukair Shetha A, Duncan Kristina M, Harris Christopher W, Belyavskaya Elena, Sternberg Esther M
Section on Neuroendocrine Immunology and Behavior, National Institute of Mental Health/NIH, Bethesda, Maryland, USA.
Nucl Recept Signal. 2007 Aug 3;5:e007. doi: 10.1621/nrs.05007.
Several methods are currently employed to evaluate expression of steroid hormone receptors in tissues and cells, including real-time reverse-transcriptase polymerase chain reaction (real-time RT-PCR) and western blot assays. These methods require homogenization of cells, thereby preventing evaluation of individual cells or specific cell types in a given tissue sample. In addition, methods such as real-time RT-PCR assess mRNA levels, which may be subject to posttranslational modifications that prevent subsequent production of functional proteins. Flow cytometry is a fluorescence-based technique commonly used to evaluate expression of cell surface and intracellular proteins. This method is especially useful as it allows for single-cell analysis and can be utilized to determine the amount of receptor expressed by individual cells. Flow cytometry is commonly used to analyze immune cell activity and determine functionality based on changes in expression of cell surface molecules, as well as intracellular proteins (such as cytokines). Here, we describe a method to identify protein expression of steroid hormone receptors by rat leukocytes from different organs (spleen, liver and thymus) using flow cytometry. We examined expression of glucocorticoid receptor (GR), androgen receptor (AR) and progesterone receptor (PR) by cells at these sites and were able to demonstrate expression of receptors, as well as the intensity of expression of each receptor. This method is useful for rapid, high throughput measurement of steroid hormone receptors at the protein level in single, intact cells and would be valuable to determine which cells are more likely to respond to steroid hormone treatment.
目前采用多种方法来评估组织和细胞中类固醇激素受体的表达,包括实时逆转录聚合酶链反应(实时RT-PCR)和蛋白质免疫印迹分析。这些方法需要对细胞进行匀浆处理,从而无法对给定组织样本中的单个细胞或特定细胞类型进行评估。此外,实时RT-PCR等方法评估的是mRNA水平,而mRNA可能会受到翻译后修饰的影响,从而阻止后续功能性蛋白质的产生。流式细胞术是一种基于荧光的技术,常用于评估细胞表面和细胞内蛋白质的表达。这种方法特别有用,因为它允许进行单细胞分析,并可用于确定单个细胞表达的受体数量。流式细胞术通常用于分析免疫细胞活性,并根据细胞表面分子以及细胞内蛋白质(如细胞因子)表达的变化来确定其功能。在这里,我们描述了一种使用流式细胞术从大鼠不同器官(脾脏、肝脏和胸腺)的白细胞中鉴定类固醇激素受体蛋白表达的方法。我们检测了这些部位细胞中糖皮质激素受体(GR)、雄激素受体(AR)和孕激素受体(PR)的表达,并能够证明受体的表达以及每种受体的表达强度。这种方法对于在单个完整细胞中快速、高通量地测量蛋白质水平的类固醇激素受体很有用,并且对于确定哪些细胞更有可能对类固醇激素治疗产生反应具有重要价值。