ANTIEPILEPTIC EFFECT OF GENETICALLY MODIFIED FIBROBLASTS

转基因成纤维细胞的抗癫痫作用

• 批准号: 3084432
• 负责人: LAN S CHEN
• 金额: $ 9.13万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 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 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3084432
• 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; microorganism genetics; nervous system transplantation; plasmids; reporter genes; secretion; transfection; transfection /expression vector; 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生科学家 致力于神经科学 最终的方向是发展改良型 治疗人癫痫的方法。 申请人计划使用 结合基因转移和神经移植作为一种工具，研究 癫痫发作传播和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 氨基转移酶抑制剂（如γ-乙烯基GABA）进入离散的大脑区域 (e.g.黑质、脚内核、杏仁核和下丘脑 丘）在各种癫痫动物模型中均能抑制癫痫发作。 此外，GABA局部慢性输注到运动皮层完全， 自然阻断行为和电图癫痫发作 感光狒狒 血脑屏障的不通透性， 然而，由于这些药物的全身毒性， 抗癫痫药 因此，本建议的总体目标是 来测试移植转基因的，分泌GABA的 成纤维细胞进入离散的脑区域可以改变癫痫发作的表现。 为了解决这一目标，谷氨酸脱羧酶（GAD）基因将被克隆。 作为报告基因，逆转录病毒将作为基因递送载体 并且原代皮肤成纤维细胞和成纤维细胞细胞系都将是 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 容易获得且易于体外培养和操作;（3） 潜在治疗的长期策略的关键是使用 原代皮肤成纤维细胞作为自体移植的供体细胞。 具体而言，将分离野生型逆转录病毒的前病毒基因 通过替换病毒所必需的基因来构建质粒， GAD基因的复制。 可传播但可复制- 携带GAD基因的不合格逆转录病毒载体可以通过以下方法产生： 将所构建的质粒引入含有所述质粒的细胞系中， 逆转录病毒辅助病毒的前病毒基因。 通过转染 载体，GAD基因将整合到成纤维细胞中。 体外 GAD基因的表达将通过测量mRNA来定量，并分泌 GABA 为了监测体内功能， 将成纤维细胞移植到海马或侧脑室中， 测试对癫痫发作现象的影响（行为和 癫痫发作）、癫痫发作阈值和癫痫诱发的 神经元变性 用于这些目的的模型系统是一种慢性病 穹窿海马伞损伤诱导的癫痫模型和 杏仁核内注射红藻氨酸诱发边缘癫痫发作。 的 移植物长期存活、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的抗体。