ANTIEPILEPTIC EFFECT OF GENETICALLY MODIFIED FIBROBLASTS

转基因成纤维细胞的抗癫痫作用

• 批准号: 3084431
• 负责人: LAN S CHEN
• 金额: $ 3.84万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1992-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3084431
• 关键词: GABA receptor; Retroviridae; biomarker; drug delivery systems; electroencephalography; fibroblasts; gamma aminobutyrate; gene expression; generalized seizures; genetic manipulation; glutamate decarboxylase; graft versus host disease; hippocampus; histochemistry /cytochemistry; immunochemistry; messenger RNA; nervous system transplantation; plasmids; reporter genes; secretion; transfection; transposon /insertion element

The applicant's long term career-goal is to become a physician scientist dedicated to neuroscience. The ultimate direction is to develop improved methods of treating human epilepsy. The applicant plans to use a combination of gene transfer and neural grafting as a tool to study seizure propagation and as a potential therapeutic approach to epilepsy. Local application of GABA receptor agonist (e.g. muscimol) or GABA transaminase inhibitor (e.g. gamma-vinyl GABA) into discrete brain regions (e.g. substantia nigra, entopeduncular nucleus, amygdala and inferior colliculus) could suppress seizure in various animal models of epilepsy. Moreover, chronic infusion of GABA locally into motor cortex completely blocked the behavioral and electrographical seizure in naturally photosensitive baboon. The impermeability to blood-brain-barrier and global toxicity, however, preclude the systemic use of these agents as antiepileptic drugs. Therefore, the overall objective of this proposal is to test the idea that grafting the genetically modified, GABA-secreting fibroblasts into discrete brain regions can modify seizure manifestations. To address this objective, glutamic acid decarboxylase (GAD) gene will be used as the reporter gene, retroviruses will be the gene delivery vectors and both primary skin fibroblasts and fibroblasts cell lines will be the donor cells. The reasons for the use of fibroblasts as donor cells are (1) retroviruses require dividing cells for infection; (2) fibroblasts are readily available and easily cultured and manipulated in vitro; (3) essential to the long-term strategy of potential therapy is the use of primary skin fibroblasts as donor cells for autograft. Specifically, the proviral gene of wild-type retrovirus will be isolated to construct the plasmid by replacing the genes necessary for viral replication with the GAD gene. The transmissible but replication- incompetent retroviral vectors carrying GAD gene can be produced by introducing the constructed plasmid to the cells lines containing the proviral gene of retroviral helper virus. By transfection with the vectors, GAD gene will be integrated into fibroblasts. The in vitro expression of GAD gene will be quantitated by measuring mRNA and secreted GABA. To monitor the function in vivo, the genetically modified fibroblasts will be grafted into hippocampus or lateral ventricle and tested for the effects on the seizure phenomenon (behavioral and electroencphalographical seizure), seizure threshold and seizure-induced neuronal degeneration. The model systems for these purposes are a chronic model of epilepsy induced by fimbria-fornix lesions and an acute model of limbic seizure induced by intraamygdala injection of kainic acid. The long-term graft survival, in vitro gene expression and host immune reaction will be assessed by immunohistochemical approaches using antibodies to various specific cell markers.

申请人的长期职业目标是成为一名医师科学家 致力于神经科学。 最终的方向是开发改进 治疗人类癫痫的方法。 申请人计划使用 基因转移和神经移植相结合作为研究工具 癫痫发作传播并作为癫痫的潜在治疗方法。 局部应用 GABA 受体激动剂（例如蝇蕈醇）或 GABA 将转氨酶抑制剂（例如 γ-乙烯基 GABA）注入离散的大脑区域 （例如黑质、内脚核、杏仁核和下核） colliculus）可以抑制各种癫痫动物模型的癫痫发作。 此外，长期将 GABA 完全局部注入运动皮层 自然地阻止行为和电图癫痫发作 感光狒狒。 血脑屏障的不通透性和 然而，全身毒性阻碍了这些药物的全身使用，因为 抗癫痫药物。 因此，本提案的总体目标是 测试嫁接转基因、分泌 GABA 的想法 成纤维细胞进入离散的大脑区域可以改变癫痫发作的表现。 为了实现这一目标，谷氨酸脱羧酶（GAD）基因将被 逆转录病毒作为报告基因，将成为基因传递载体 原代皮肤成纤维细胞和成纤维细胞系都将是 供体细胞。 使用成纤维细胞作为供体细胞的原因是 (1)逆转录病毒需要分裂细胞进行感染； (2)成纤维细胞是 易于获得且易于体外培养和操作； (3) 潜在疗法的长期策略至关重要的是使用 原代皮肤成纤维细胞作为自体移植的供体细胞。 具体地，将分离野生型逆转录病毒的前病毒基因 通过替换病毒所需的基因来构建质粒 与GAD基因的复制。 可传播但可复制- 携带GAD基因的无活性逆转录病毒载体可以通过以下方式产生 将构建的质粒导入含有 逆转录病毒辅助病毒的前病毒基因。 通过转染 载体中，GAD基因将整合到成纤维细胞中。 体外 通过测量 mRNA 来定量 GAD 基因的表达并分泌 伽马氨基丁酸。 为了监测体内功能，转基因 成纤维细胞将被移植到海马或侧脑室 测试对癫痫现象的影响（行为和 脑电图癫痫发作）、癫痫阈值和癫痫诱发 神经元变性。 用于这些目的的模型系统是一个长期的 穹窿伞损伤诱发的癫痫模型和急性癫痫模型 杏仁核内注射红藻氨酸诱导边缘癫痫发作。 这 长期移植物存活、体外基因表达和宿主免疫 将通过免疫组织化学方法评估反应 针对各种特定细胞标记物的抗体。