Ignatowski T A, Bidlack J M
Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, New York, USA.
J Pharmacol Exp Ther. 1998 Jan;284(1):298-306.
Recent studies have shown kappa opioid receptor labeling on the R1EGO thymoma cell line by indirect immunofluorescence and flow cytometric analysis. The present study used a fluorescein-labeled arylacetamide (FITC-AA), a kappa opioid ligand, in conjunction with biotin-conjugated anti-fluorescein IgG and extravidin-R-phycoerythrin (PE), along with double-labeling with antibodies against specific immune cell surface markers to determine which subpopulation(s) of thymocytes express the kappa opioid receptor. Thymocytes, isolated from 6- to 8-week-old C57BL/6ByJ mice, incubated with FITC-AA followed by the PE amplification procedure, demonstrated labeling of the kappa opioid receptor. This labeling was inhibited 55 +/- 4% above background by excess nor-binaltorphimine (nor-BNI), a kappa selective antagonist. This kappa opioid receptor positive population consisted of 58 +/- 2% of all gated thymocytes. Phenotypic characterization determined that not only were 64 +/- 3% of the gated thymocytes CD4+/kappa opioid receptor positive, but 60 +/- 1% of all thymocytes were CD8+/kappa opioid receptor positive. Two subpopulations of CD3+ thymocytes, consisting of both mature and immature cells, also displayed labeling for the kappa opioid receptor. Double-labeling of thymocytes with anti-CD4 and anti-CD8 antibodies demonstrated 82 +/- 0.5% of these cells were of the double-positive phenotype. Therefore, these findings demonstrate that the thymocytes, which express the kappa opioid receptor, are predominantly of the immature CD4+/CD8+ phenotype. Collectively, these findings not only establish the presence of the kappa opioid receptor on immune cells involved in opioid responsiveness, but further indicate that this technique allows for the identification of distinct lymphocyte subpopulations which express the receptor.
最近的研究通过间接免疫荧光和流式细胞术分析显示，κ阿片受体在R1EGO胸腺瘤细胞系上有标记。本研究使用了一种荧光素标记的芳基乙酰胺（FITC - AA），一种κ阿片配体，结合生物素偶联的抗荧光素IgG和抗生物素蛋白 - R - 藻红蛋白（PE），并与针对特定免疫细胞表面标志物的抗体进行双重标记，以确定哪些胸腺细胞亚群表达κ阿片受体。从6至8周龄的C57BL / 6ByJ小鼠中分离出胸腺细胞，用FITC - AA孵育，然后进行PE扩增程序，结果显示有κ阿片受体的标记。这种标记被过量的κ选择性拮抗剂去甲 - 纳洛酮啡烷（nor - BNI）抑制，比背景高55±4%。这个κ阿片受体阳性群体占所有门控胸腺细胞的58±2%。表型特征确定，不仅64±3%的门控胸腺细胞是CD4 + /κ阿片受体阳性，而且所有胸腺细胞中有60±1%是CD8 + /κ阿片受体阳性。由成熟和未成熟细胞组成的CD3 + 胸腺细胞的两个亚群也显示有κ阿片受体的标记。用抗CD4和抗CD8抗体对胸腺细胞进行双重标记显示，这些细胞中有82±0.5%是双阳性表型。因此，这些发现表明，表达κ阿片受体的胸腺细胞主要是未成熟的CD4 + /CD8 + 表型。总的来说，这些发现不仅证实了在参与阿片反应的免疫细胞上存在κ阿片受体，而且进一步表明这种技术能够识别表达该受体的不同淋巴细胞亚群。
