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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，通过用cDNA转染已知的细胞系来开发表达重要蛋白质如受体、酶、离子通道等的细胞模型，用于建立有用的系统来评估药物功效和创造新药。换句话说，建立稳定的细胞系是有用且重要的，其中功能蛋白质不是内源性存在的，而是过表达的。最近，我们研究了原代培养物和已建立的细胞系中CaM激酶II的激活和自磷酸化，以响应细胞外刺激，例如神经递质、本研究报道了在LTP诱导过程中海马CA 1区CaM激酶Ⅱ的激活。1）用合成的引物，通过PCR方法制备了CaM激酶Ⅱ α、β、γ、δ亚基的cDNA。将cDNA插入表达载体pCAGGSneo中。通过电穿孔法或脂质体法将cDNA转染PC 12细胞或NG 108 -15细胞。使用其中仅插入载体而没有CaM激酶II亚基同种型的cDNA的模拟细胞。2)制备稳定的转染有CaM激酶II α cDNA的PC 12细胞。3)稳定细胞系中CaM激酶Ⅱ α的表达可抑制二丁酰cAMP诱导的神经突起生长。4）免疫家兔制备特异性抗CaM激酶Ⅱ δ抗体。5)用PCR方法制备CaM激酶Ⅱ δ亚基各亚型的cDNA并测序。CaM激酶Ⅱ δ的三种亚型均具有核定位信号。6)大鼠小脑矢状面免疫组织化学染色结果表明，CaM激酶Ⅱ δ定位于颗粒细胞核，而浦肯野细胞核内未见CaM激酶Ⅱ δ的表达。