Protection of Neuronal Death by Induction of Glutamate Transporter Gene

谷氨酸转运蛋白基因诱导保护神经元死亡

• 批准号: 09557119
• 负责人: KONDOH Takeshi
• 金额: $ 7.81万
• 依托单位: Kobe University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09557119/
• 关键词: In vivo gene transfer; Neuronal Transplantation; Blood-brain Barrier; GFP; HUVEC; Electroporation

Gene transfer by electroporation was attempted in the normal brain. The reporter gene pEGFP-C1(25g/5μl) was injected in the striatum of young adult rats and various ranges of square electrical impulses were applied by using a pair of electrodes that were placed in the striatum. After five days, histological examination revealed that the impulses of high voltage caused extensive tissue damage whereas impulses of lower range (200-400mJ) resulted in the transfection of more than 300 cells per brain, which were widely distributed in the subependymal region of the lateral ventricle and extended long processes into the striatum.Human umbilical vein endothelial cells (HUVECs) were transplanted in athymic mouse brain and neovascularization of grafted endothelial cells was studied. HUVECa were transfected by a reporter gene pEGFPE-N1 in vitro and grafted stereotactically in unilateral striatum of adult nude mice. Histological studies in four weeks revealed that grafted HUVECs newly formed micro … More vessels in brain, which were migrated and fused with host vessels. Intravenous injection of Evans Blue prior to sacrificing animals resulted in no extravasation of dye, indicating that'a blood-brain barrier was formed by the grafted HUVECs. Immunohistochemistry demonstrated that host astrocytes extended glial feet on the grafted endothelial cells and a part of the newly formed vessels were positive with glucose transporter-1. These results indicate that endothelial cells from an ectopic origin have the potential to from a blood-brain barrier after grafting in the central nervous system.Neuronal progenitor cells have been widly studied with the purpose of regeneration of injured central nervous system. For the grafting of these cells, not pure single cell suspension of stem cells but spheres composed with progenitor cells have been reported to demonstrate improved survival after grafting. In this study, rat neuronal stem cells were obtained from E-14 rat subventriclur zone followed by free floating culture in EGF-containing medium as previously reported by Weiss et al. After four passage in four weeks, single stem cells were left to grow without dissociation for two months by changing the medium weekly. The spheres became 500-800 um in diameter and then preserved in Hibernation Medium E (Gibco BRL) for a week at 4℃. Those sphere were labeled by PHK26 right before the grafting. Two different type of cerebral ischemia has been prepared in host adult rats. One is middle cerebral artery occulusion by phtochemical method and the other is endothelin injection (ET-1 ; 0.05ng/animal) into unilateral striatum. One week following transplantation of hibernated neuroprogenitor sphere demonstrated (A) no graft survival in the core of ischemic lesion in MCA occlusion model, (B) survival of small clusters in the border of ischemic core demonstrated by faint fluorscent marker, (C) dense clusters and migrating cells in the most brain in endothelin-injected striatum. Immunostaining using anti-MAP-2 and anti-GFAP serum demonstrated neuronal as well as glial cells in the grafts. Cryopreservation in DMSO-containing medium at-180℃or preservation in hibernation medium longer than one week demonstrated flagile sphere which were unable to handle for grafting surgery. This study demonstrated that neuronal progenitor cell sphere are able to survive in the brain after preservation for one week. Less

在正常大脑中，曾尝试通过电穿孔进行基因转移。将报告基因pEGFP-c1(25g/5μL)注入幼年大鼠纹状体内，用放置在纹状体内的一对电极施加不同范围的方波电脉冲。5天后，组织学检查显示，高电压冲击造成广泛的组织损伤，而低电压(200-400mJ)的冲击导致每个脑内300多个细胞被转移，这些细胞广泛分布在侧脑室管膜下，并向纹状体延伸长突起。将人脐静脉内皮细胞移植到无菌小鼠脑内，并对移植的内皮细胞进行新生血管的研究。体外将报告基因pEGFPE-N1导入HUVECa，立体定向移植于成年裸鼠单侧纹状体内。四周的组织学研究表明，移植的人脐静脉内皮细胞新形成了微型…脑内血管增多，迁移后与宿主血管融合。在处死动物之前静脉注射伊文思蓝没有引起染料外渗，这表明移植的人脐静脉内皮细胞形成了血脑屏障。免疫组织化学显示宿主星形胶质细胞在移植的内皮细胞上延伸胶质足，部分新生血管葡萄糖转运蛋白-1阳性。这些结果表明，异位来源的内皮细胞在移植到中枢神经系统后具有形成血脑屏障的潜力。神经前体细胞已被广泛研究，目的是为了再生受损的中枢神经系统。对于这些细胞的移植，据报道，不是单纯的干细胞单细胞悬浮液，而是由祖细胞组成的球体显示出移植后存活率的提高。在这项研究中，从E-14大鼠脑室下区获得了大鼠神经元干细胞，然后在含有EGF的培养液中进行自由漂浮培养，这是Weiss等人以前报道的。在4周内传代4次后，通过每周更换培养液，使单个干细胞在不分离的情况下生长2个月。球体直径为500~800um，在冬眠培养液E(Gibco BRL)中4℃保存一周。这些球体在移植前被PHK26标记。在宿主成年大鼠中制备了两种不同类型的脑缺血模型。一种是光化学法阻断大脑中动脉，另一种是单侧纹状体内注射内皮素(ET-1，0.05 ng/只)。移植冬眠神经前体细胞球1周后发现：(A)大脑中动脉闭塞模型缺血灶中心无移植物存活，(B)微弱荧光标记物显示缺血灶边缘有小簇存活，(C)内皮素注射纹状体大部分脑区有致密的簇状细胞和迁移细胞。用抗MAP-2和抗GFAP血清进行免疫组织化学染色，可见移植物内有神经细胞和胶质细胞。在-180℃含二甲基亚砜的培养液中冷冻保存或在冬眠介质中保存一周以上，均可见脆弱的球体，不能用于移植手术。本研究证明神经元祖细胞球在保存一周后仍能在脑内存活。较少

项目成果

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