永生化神经前体细胞膜表面μ阿片受体表达的鉴定

Identification ofμopioid receptor expression in immortalized neural progenitor cells Abstract

目的:阿片类物质可调控神经细胞的增殖和分化,为进一步明确阿片类物质对神经前体细胞的影响,对永生化神经前体细胞膜表面μ阿片受体的表达进行鉴定。方法:实验于2007-04/06在华中科技大学同济医学院附属同济医院麻醉科实验室完成。通过反转录-聚合酶链反应及免疫荧光化学技术,分别从mRNA水平及蛋白质水平直接检测永生化神经前体细胞膜表面μ阿片受体的表达。另外将永生化神经前体细胞分别予以μ阿片受体激动剂吗啡及非选择性阿片受体拮抗剂纳洛酮处理,通过激光扫描共聚焦显微镜定量检测加药前、后永生化神经前体细胞内钙离子浓度(intracellular calcium concentration,[Ca^(2+)]_i)的改变,从而间接鉴定永生化神经前体细胞膜表面μ阿片受体的表达。结果:反转录-聚合酶链反应结果表明永生化神经前体细胞内有μ阿片受体mRNA存在,免疫荧光化学结果显示μ受体主要定位表达于永生化神经前体细胞细胞膜及细胞浆。受吗啡刺激后,永生化神经前体细胞内[Ca^(2+)]_i较刺激前显著增高(t=7.61,P<0.05);加入纳洛酮后[Ca^(2+)]_i亦呈显著增高趋势(t=4.96,P<0.05);待纳洛酮作用平稳后再加入吗啡,[Ca^(2+)]_i无明显改变(t=1.00,P>0.05)。结论:永生化神经前体细胞膜表面确有μ阿片类受体表达,这为从体外水平研究阿片类物质对神经前体细胞增殖及分化的影响提供了一种易于培养、稳定可靠的工具细胞系。

AIM： Opioid could regulate the proliferation and differentiation of nerve cells. To further confirm the effect of opioid on neural progenitor cell, the μ opioid receptor expression on the cytomembrane of immortalized neural progenitor cell （INPC） was identified. METHODS： The experiment was conducted in the laboratory of Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from April to June 2007. μ opioid receptor expression of mRNA and protein levels in the INPC was determined directly by RT-PCR and immunofluorescence, respectively. In addition, with the calcium-sensitive fluorescent dye Fluo4/AM, intracellular calcium concentration （[Ca^2＋]i） of INPC was analyzed by the Laser Scanning Confocal Microscope after the presence of morphine （μ opioid receptor agonist） and naloxone （μ opioid receptor non-selective antagonist）. Therefore, μ opioid receptor expression was identified indirectly by this function test. RESULTS： The results of RT-PCR revealed that there was mRNA of μopioid receptor produced by this cell line. Accordingly, the results of immunofluorescence displayed that μ opioid receptors were mostly located both in the cytomembrane and cytoplasm. After the stimulation of morphine, [Ca^2＋]i was increased significantly temporarily （t =7.61, P 〈 0.05）. On the other hand, [Ca^2＋]i also presented a temporal upward trends when given naloxone （t =4.96, P 〈 0.05）. However, when the action of naloxone faded, [Ca^2＋]i was stable although at the presence of morphine （t =1 .00, P 〉 0.05）. CONCLUSION： μ opioid receptor is expressed in the cytoplasmic membrane of INPC. So this cell line is a very useful tool for the in vitro research on the effects of opioid on neural progenitor cells.