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

单纯疱疹病毒(HSV-L，这里称为“HSV”)载体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传递到细胞。我们首先改进和完善了利用有缺陷的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。NMDA谷氨酸受体通道由两个(epsilon，Zeta)亚家族组成，功能变异基于epsilon家族亚单位的分子多样性。接下来，我们构建了含有zeta1基因的缺陷型HSV载体，通过RT-PCR、斑点杂交和Western印迹分析，证实该HSV克隆在兔皮肤细胞中有高水平的表达。时程研究表明，重组Zeta1蛋白在感染后3h首次检测到，并在感染后24~48h达到最大。利用杆状病毒载体对谷氨酸受体通道的配体结合区进行了分子分析。进一步，将缺陷HSV载体构建成表达小鼠干扰素-γ的载体，用于RSV小鼠胶质瘤的实验性基因治疗。免疫组织化学方法检测小鼠体内和体外干扰素-γ的表达。该工程载体在体外可抑制RSV胶质瘤的增殖，其瘤内局部注射可引起瘤内坏死。在RSV小鼠胶质瘤移植模型中检测该载体的免疫学效应。转移到细胞中的干扰素-γ基因具有肿瘤免疫的作用，这可能与自然杀伤细胞有关。这些结果表明，表达干扰素-γ的工程HSV载体可能通过瘤内注射或外源性肿瘤疫苗用于恶性胶质瘤的基因治疗。

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.