Micro-quantification of cell-cycle regulating factors by Enzyme-Linked Immuno-Cycling Assay

神经细胞必须在脱离细胞周期后发育成熟，不再分裂。尽管如此，细胞周期调控因子，即细胞周期蛋白和细胞周期蛋白依赖性激酶（cdk）被证明存在于有丝分裂后的神经元中。为了阐明这些分子在神经元中的生理作用，我们首先在鸡胚视网膜中鉴定了细胞周期蛋白和cdk的分子种类。使用培养的视网膜神经元的免疫组织化学研究显示，针对细胞周期蛋白D1或cdk 5的抗体对包括未成熟的感光细胞在内的所有种类的视网膜神经元均呈免疫反应性。然后用免疫印迹法检测大鼠小脑中cyclin D1和cdk 5表达的发育性变化。结果发现，cyclin D1在出生后至成年期一直表达，而cdk 5在8日龄左右表达短暂，此时小脑层的形成非常显著。这些结果提示cdk 5可能参与了小脑颗粒细胞的成熟。我们还发现，一氧化氮（NO）是必要的成熟和迁移的颗粒细胞在层形成的小脑。当一氧化氮合酶（NOS）被不可逆地抑制，迁移的颗粒细胞急剧下降，从而扰动层的形成发生。为了鉴定引起这种现象的NOS亚类，将约1 - 3 μ g干重的来自外部颗粒层、分子层和内部颗粒层的组织进行显微镜下观察并通过微量免疫印迹进行分析。显微分析显示，在成层过程中，神经元型NOS和内皮型NOS在各层均有表达。

Neural cells must develop to mature after free from cell-cycle and never divide again. Despite these facts, cell-cycle regulating factors, i.e.cyclins and cyclin-dependent kinases (cdk) are shown to be exist in post-mitotic neurons. To clarify the physiological roles of those molecules in neuron, we first intended to identify the molecular species of cyclin and cdk in embryonic chick retina. The immunohistochemical studies using cultured retinal neurons revealed that antibodies raised against cyclin D1 or cdk5 were immunoreactive to all the kind of retinal neurons including immature photoreceptor cells. And then next we examined developmental change of cyclin D1 and cdk5 expression in rat cerebellum by immunoblotting. It was found that cyclin D1 was consistently expressed after birth to adulthood, however expression of cdk5 was transient around 8-days-old where the layr formation of cerebellum is extremely remarkable. These results suggest that cdk5 may be involved in the maturation of granule cells in cerebellum. We also found that nitric oxide (NO) is necessary for the maturation and migration of granule cells during the layr formation of the cerebellum. When nitric oxide synthase (NOS) was irreversibly inhibited, migration of granule cells was drastically decreased, and consequently perturbation of the layr formation occurred. To identify the subclass of NOS responsible for this phenomenon, about 1 to 3 mug in dry weight of tissue from external granular layrs, molecular layrs and internal granular layrs was electrophoresed and analyzed by micro-immunoblotting. Micro-analysis revealed that neuronal-type NOS and also endothelial-type NOS were expressed in each layrs during the layr formation.