Molecular analysis of gene expression and its regulation of glutamate receptor.

近年来，将外源基因直接导入哺乳动物大脑和培养的神经元的分子神经生物学方法已被开发用于神经科学研究和神经系统疾病的基因治疗。单纯疱疹病毒1 （HSV-1，在这里称为“HSV”）是一种嗜神经病毒，有望成为候选的病毒载体之一。我们首先改进和完善了利用缺陷HSV载体进行基因传递的方法，并总结了HSV表达系统的实验方案。接下来，我们使用表达系统分析谷氨酸受体（GluR）通道。NMDA谷氨酸受体通道由两个（epsilon, zeta）亚家族组成，功能变化基于epsilon家族亚基的分子多样性。在本研究中，我们构建了在CMV启动子下含有zeta1， epsilon1或epsilon2 cDNA的缺陷HSV载体。通过RT-PCR、dot blot和Western blot分析，证实了各HSV克隆感染兔皮肤细胞中高水平表达。我们分析了在Vero和CHO细胞中表达的重组受体的分子和功能特性，并进行了体内实验。此外，我们构建了zeta1亚基缺失突变体或ampa选择性GluRalpha1亚基突变体的杆状病毒克隆，并对这些蛋白的配体结合区进行了详细分析。我们还构建了干扰素- γ、白细胞介素2或参与抑制神经元细胞死亡的未知因子的HSV克隆，并利用这些HSV载体进行了体外和体内实验。

Recently, molecular neurobiological approaches for introducing foreign genes directly into mammalian brains as well as into cultured neurons have been being developed for neuroscience researches and for gene therapy of neurological disorders. Herpes simplex virus 1 (HSV-1 ; termed "HSV" here), which is a neurotropic virus, has been expected to be one of the viral vector candidates for the purposes. We first improved and refined the methodology of gene delivery using defective HSV vectors, and summarized the experimental protocol for the HSV expression system. Next we analyzed glutamate receptor (GluR) channels using the expression system. NMDA glutamate receptor channels consist of the two (epsilon, zeta) subfamilies and functional variations are based on the molecular multiplicity of epsilon family subunits. In this study, we constructed the defective HSV vectors, which contain zeta1, epsilon1 or epsilon2 cDNA under the CMV promoter. High-level expression was confirmed in the rabbit skin cells infected with each HSV clone by using RT-PCR,dot blot and Western blot analyzes. We have analyzed the molecular and functional properties of recombinant receptors expressed in Vero and CHO cells, and processed in in vivo experments. Further, we constructed baculovirus clones of deleted mutants of zeta1 subunit or site-directed mutants of AMPA-selective GluRalpha1 subunit proteins, and proceeded the detailed analysis of ligand binding regions of the proteins. We also constructed HSV clones of interferon-gamma, interleukine 2, or unknown factors involved in inhibition of neuronal cell death, and in vitro and in vivo experiments using these HSV vectors have been in progress.