Gene expression analyses of glutamate receptor channels by using viral vectors.

单纯疱疹病毒（HSV-1，在此称为“HSV”）载体具有在神经元中建立长期无毒潜伏期的倾向，并且病毒很大，允许几个基因插入并递送至细胞。我们首先改进和完善了缺陷型HSV载体的基因递送方法，并总结了HSV表达系统的实验方案。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。构建了含zeta 1基因的HSV缺陷型表达载体，经RT-PCR、斑点杂交和Western blot分析，证实zeta 1基因在感染的兔皮肤细胞中得到高效表达。时程分析表明，重组zeta 1蛋白在感染后3 h首次出现，在感染后24-48 h达到高峰。谷氨酸受体通道的配体结合域的分子分析也进行了使用杆状病毒载体。此外，将缺陷型HSV载体工程化以表达小鼠干扰素（IFN）-γ，用于RSV小鼠胶质瘤的实验性基因治疗。小鼠IFN-γ的表达在体外和体内用免疫组织化学显示。该工程载体在体外抑制RSV胶质瘤的增殖，并且其瘤内局部注射在体内引起瘤内坏死。在RSV小鼠胶质瘤移植模型中检测该载体的免疫效果。IFN-γ基因转染细胞后具有肿瘤疫苗效应，提示其与自然杀伤细胞有关。这些结果表明，表达IFN-γ基因的HSV载体可用于肿瘤局部注射或外源性肿瘤疫苗的基因治疗。

The herpes simplex viral (HSV-l, termed "HSV" here) vectors have the propensity to establish long-term non-toxic latency in neurons, and the virus is large, allowing several genes to be inserted and delivered to cells. We first improved and refined the methodology of gene delivery using defective HSV vectors, and summarized the experimental protocols for the HSV 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. Next, we constructed the defective HSV vectors which contain zeta1 cDNA.High-level expression was confirmed in the rabbit skin cells infected with the HSV clone by using RT- PCR, dot blot and Western blot analyses. Time-course study indicated that the recombinant zeta1 protein (125 kDa) was detected for the first time at 3 h postinfection, and reached maximum at 24-48 h postinfection. Molecular analyses of ligand-binding domains of glutamate receptor channels were also proceeded by using baculovirus vectors. Further, defective HSV vector was engineered to express mouse interferon (IFN)-gamma for experimental gene therapy for RSV mouse glioma. Expression of mouse IFN-gamma was shown in vitro and in vivo with immunohistochemistry. This engineered vector inhibited proliferation of RSV glioma in vitro, and its intratumoral local injection caused intratumoral necrosis in vivo. The immunological effect of this vector was examined in RSV mouse glioma implantation model. The IFN-gamma gene transferred to the cells had the effect of tumor vaccination, which was suggested to be related to natural killer cellsr These results revealed that the engineered HSV vector to express IFN-gamma may be useful for gene therapy of malignant glioma through either intratumoral local injection or exogenous tumor vaccination.