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疾病的基因治疗。单纯疱疹病毒1（HSV-1 ;在此称为“HSV”）是一种嗜神经性病毒，预期是用于该目的的候选病毒载体之一。本文首先对缺陷型HSV载体的基因转染方法进行了改进和完善，并对HSV表达系统的实验方案进行了总结。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（β，ζ）亚家族组成，并且功能变化基于β家族亚基的分子多样性。在这项研究中，我们构建了缺陷型HSV载体，其中包含zeta 1，ε 1或ε 2的CMV启动子下的cDNA。经RT-PCR、斑点杂交和Western印迹分析证实，各HSV克隆在感染的兔皮肤细胞中均有高水平表达。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zeta 1亚基缺失突变体或AMPA选择性GluRalpha 1亚基蛋白定点突变体的杆状病毒克隆，并对蛋白质的配体结合区域进行了详细分析。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，并且使用这些HSV载体的体外和体内实验已经在进行中。

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.