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Establishment of cell models on transfection of functional protein and the study on brain signal transduction

功能蛋白转染细胞模型的建立及脑信号转导研究

基本信息

批准号：
12557011
负责人：
MIYAMOTO Eishichi
金额：
$ 8.58万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557011/
关键词：
CaM kivases gene expression Ca^<2+> signaling dopamine D2 receptor nuclear localization signal NG108--15 cell brain-derived neurotrophic factor cell model CaMキナーゼII CaMキナーゼIIサブタイプ

项目摘要

The present study intends to establish the model of the cell line in which functionally important proteins such as receptors, enzymes, ion channels are expressed. Stimulation of the established cells with neurotransmitters hormones, growth factors etc. activates the expressed proteins in the cells. These models serve as the established systems to analyze effects of drugs and to create new drugs.The study focuses the analysis on the intracellular calcium (Ca^<2+>) and on the functional significance of CaM kinases I, II, III, IV and kinase. The cytosolic and nuclear isoforms of CaM kinase II were expressed in NG108-15 cells in which the expression of brain-derived neurotrophic factor (BDNF) was examined. The expression of BDNF was not increased by the transfection of the cytosolic isoforms such as CaM kinase II δ1, and δ2 but was increased by CaM kinase II δ3 and αB. The quantitative method of RT-PCR for the amount of the mRNA indicated the increase in Exon IV-BDNF mRNA but not Exon III-BDNF mRNA. This suggest that the nuclear isoforms only increase the mRNA and proteins of BDNF.Dopamine D2 receptors (D2R) consist of the long chain receptor (D2RL) and the short chain receptor (D2RS). Both cDNAs were transfected into NG108-15 cells and the stable cells with the expression of each receptor were obtained. The analysis by confocal laser microscopy revealed different localization of each receptor in the cells. Stimulation of D2R increased the concentration of the intracellular Ca^<2+>. The cDNA of CaM kinase II δ3 with nuclear localization signal was transiently transfected in the stable cells of D2RL. Stimulation of D2R of the transfected cells with the agonist resulted in activation of the nuclear isoform of CaM kinase II and the increase in the expression of BDNF.

本研究旨在建立具有重要功能的蛋白质如受体、酶、离子通道等在细胞系中表达的模型。用神经递质、激素、生长因子等刺激已建立的细胞，激活细胞中表达的蛋白质。这些模型作为既定的系统来分析药物的效果和创造新的药物。本研究重点分析细胞内钙（Ca^<2+>）和CaM激酶I、II、III、IV和激酶的功能意义。在检测脑源性神经营养因子（BDNF）表达的NG108-15细胞中表达了CaM激酶II的胞浆和核同工型。转染CaM kinase II δ1和δ2后，BDNF的表达不增加，而转染CaM kinase II δ3和αB后，BDNF的表达增加。RT-PCR定量测定mRNA量的方法显示，外显子IV-BDNF mRNA增加，而外显子III-BDNF mRNA未增加。这表明核异构体只增加BDNF的mRNA和蛋白质。多巴胺D2受体（D2R）由长链受体（D2RL）和短链受体（D2RS）组成。将这两种cdna转染到NG108-15细胞中，获得了稳定表达这两种受体的细胞。激光共聚焦显微镜分析显示，细胞中每种受体的定位不同。D2R刺激增加细胞内Ca^<2+>的浓度。在稳定的D2RL细胞中瞬时转染带有核定位信号的CaM kinase II δ3 cDNA。用激动剂刺激转染细胞的D2R导致CaM激酶II核亚型的激活和BDNF表达的增加。