Establishment of stable cells by induction of cDNAs of functional proteins and preparation of models for evaluation of drug efficacy for creation of new drugs

通过功能蛋白cDNA诱导建立稳定细胞并制备用于新药研发的药效评价模型

基本信息

批准号：
07557195
负责人：
MIYAMOTO Eishichi
金额：
$ 5.31万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

H.A.Lester reported that the development of cellular models to express important proteins such as receptors, enzymes, ion channels and so on by transfecting known cell lines with the cDNAs serves for the establishment of useful systems to evaluate drug efficacy and create new drugs. In other words, the establishment of stable cell lines in which functional proteins are not endogenously present, but overexpressed be useful and important.Recently, we have studied on activation and autophosphorylation of CaM kinase II in primary cultures and established cell lines in response to extracellular stimuli such as neurotransmitters, cell growth factors and so on. We reported the activation of CaM kinase II in the CA1 area of the hippocampus during LTP induction.The present study were carried out as follows. 1)We prepared the cDNAs of CaM kinase IIalpha, beta, gamma, delta subunits by the PCR method with synthesized primers. The cDNAs were inserted into the expression vector pCAGGSneo. PC12 cells or NG108-15 cells were transfected with the cDNAs by electropolation or the lipofectamine method. Mock cells into which only the vector is inserted without the cDNAs of CaM kinase II subunit isoforms were used. 2)Stable PC12 cells transfected with the cDNAs of CaM kinase IIalpha were prepared. 3)The expression of CaM kinase IIalpha in stable cell lines inhibited neurite outgrowth induced by dibutyryl cAMP.4)The specific antibody to CaM kinase IIdelta was prepared by immunizing rabbits. 5)The cDNAs of CaM kinase IIdelta subunit isoforms were prepared by the PCR method and sequenced. Three isoforms of CaM kinase IIdelta had nuclear localization signal. 6)The immunohistochemistry of the sagittal sections of rat cerebellum demonstrated that CaM kinase IIdelta is localized in the nuclei of granule cells, but not in the nuclei of Purkinje cells.

H.A. Lester报道说，通过使用CDNA将已知的细胞系转染已知的细胞系来建立有用的系统来评估药物疗效并创建新药，从而表达了表达重要蛋白质（例如受体，酶，离子通道等）等细胞模型的发展。换句话说，建立稳定的细胞系，其中功能蛋白不是内源性的，而是过表达的有用和重要的。非常重要的是，我们已经研究了caM激酶II在原代培养物中的激活和自磷酸化，并确定细胞系，响应细胞外刺激，以响应细胞外刺激器，例如神经递质，细胞生长因子，生长因子和其他细胞。我们报道了LTP诱导期间海马的CA1区域中CAM激酶II的激活。目前的研究如下。 1）我们通过合成引物的PCR方法准备了CAM激酶Iialpha，Beta，Gamma，Delta亚基的cDNA。将cDNA插入表达载体pcaggsneo中。 PC12细胞或NG108-15细胞通过电力或Lipofectamine方法转染CDNA。仅使用CAM激酶II亚基同工型的CYTNAS插入仅插入载体的模拟细胞。 2）制备了用CAM激酶iialpha的cDNA转染的稳定的PC12细胞。 3）CAM激酶IIALPHA在稳定的细胞系中的表达抑制了由二丁酰基cAMP诱导的神经突生长。4）与CAM激酶Iidelta的特异性抗体是通过免疫兔子来制备的。 5）CAM激酶Iidelta亚基同工型的CDNA通过PCR方法制备并进行了测序。 CAM激酶Iidelta的三种同工型具有核定位信号。 6）大鼠小脑矢状切片的免疫组织化学表明，CAM激酶IIDELTA位于颗粒细胞的核中，但不在Purkinje细胞的核中。