MOLECULAR DYNAMICS OF RECEPTORS OF LIPID SOLUBLE SIGNAL MOLECULES

将GFP-GR嵌合体载体导入培养的细胞。通过荧光素酶活性检测，证实GFP-GR嵌合体系统具有功能。地塞米松诱导的小鼠乳腺肿瘤病毒启动子(MMTV)-Luc报告基因是由地塞米松诱导的。实时成像研究表明，在缺乏配体的情况下，GFP-GR存在于细胞质中。地塞米松作用于培养细胞可诱导GFP-GR的胞核积聚，GFP-GR从胞浆向胞核的移位呈时间依赖性。地塞米松处理30min后，GFP-GR完全移位到细胞核内。核仁内未见绿色荧光。在原代培养的海马神经元和皮质胶质细胞中也观察到了同样的结果。地塞米松和皮质酮处理15~30min后，GFP-GR从核周膜的小细胞质进入细胞核，说明不同类型细胞间GR转运方式无明显差异。GFP-MR嵌合体还被导入COS-1细胞、海马神经元和皮质神经胶质细胞。就像在GFP-GR的情况下一样，在观察实时成像之前，应该验证GFP-MR嵌合体的构建在转录上是活性的。GFP-MR在大多数无配体的COS-1细胞胞浆中均有表达。MR激动剂醛固酮以时间依赖的方式诱导GFP-MR从胞浆移位到胞核。在处理后30min内，绿色荧光完全出现在细胞核内，核仁中没有GFP-MR。皮质酮处理后，COS-1细胞的细胞核中也有类似的GFP-MR积聚，核仁处没有观察到荧光。用GFP-era构建的未经雌激素处理的细胞在细胞核中显示出GFP-era。核仁中没有GFP-Era。

GFP-GR chimera construct was transfected into the cultured cells. By using luciferase assay GFP-GR chimera system was confirmed to be functional. DEX-inducible mouse mammary tumor virus promoter (MMTV)-Luc reporter was induced by DEX.Real-time imaging study has shown that in the absence of ligand GFP-GR was present in the cytoplasm of cells. Exposure of DEX to cultured cells induced the nuclear accumulation of GFP-GR and its translocation from cytoplasm-to-nucleus was time-dependent manner. Thirty min after DEX treatment GFP-GR was completely translocated into the nucleus. No green fluorescence was observed in the nucleolus. The same results were observed in the primary cultured hippocampal neurons and cortical glia. GFP-GR from the small cytoplasm of the perikarya to the nucleus was observed after 15 to 30 min after the DEX as well as corticosterone treatment, indicating that there was no conspicuous difference of trafficking manner of GR in the presence of the ligands among cell types. GFP-MR chimera construct was also transfected into COS-1 cells, hippocampal neurons, and cortical glia. As in the case of GFP-GR, GFP-MR chimera construct should be verified to be transcriptionally active before observing the real-time imaging. GFP-MR was observed in the cytoplasm of most of the COS-1 cells without the ligand. Aldosterone, an agonist for MR, induced the translocation of GFP-MR from the cytoplasm to the nucleus in a time-dependent manner. Within 30 min after the treatment green fluorescence was completely present in the nucleus and nucleolus was devoid of GFP-MR.Corticosterone treatment caused the similar accumulation of GFP-MR in the nucleus of the COS-1 cells where no fluorescence was observed at the nucleolus. Cells that were transfected by GFP-ERa construct and were not treated by estrogen showed the GFP-ERa in the nucleus. Nucleolus was devoid of GFP-ERa.