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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

基本信息

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

项目摘要

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

在正常大脑中尝试通过电穿孔进行基因转移。将报告基因pEGFP-C1（25 μ g/5μl）注射到幼年成年大鼠的纹状体内，用一对放置在纹状体内的电极施加不同幅度的方形电脉冲。5天后，组织学检查显示，高电压脉冲引起广泛的组织损伤，而低电压脉冲（200- 400 mJ）导致每个脑超过300个细胞的转染，人脐静脉内皮细胞（HUVECs）在侧脑室室管膜下广泛分布，并有长突起伸入纹状体移植于无胸腺小鼠脑内，研究移植内皮细胞的新生血管形成。将报告基因pEGFPE-N1转染人脐静脉内皮细胞（HUVECa），立体定向移植于裸鼠单侧纹状体。四周的组织学研究显示，移植的HUVEC新形成的微 ...更多信息脑内血管迁移并与宿主血管融合。在处死动物前静脉注射伊文思蓝没有导致染料外渗，表明移植的HUVECs形成了血脑屏障。免疫组化结果显示，宿主星形胶质细胞在移植的内皮细胞上延伸出胶质足，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移植到中枢神经系统后具有形成血脑屏障的潜能，神经前体细胞在中枢神经系统损伤后的再生方面已得到广泛的研究。对于这些细胞的移植，已经报道了不是干细胞的纯单细胞悬浮液，而是由祖细胞组成的球体，以证明移植后存活率的提高。在这项研究中，大鼠神经元干细胞从E-14大鼠脑室下区获得，然后在含EGF的培养基中自由漂浮培养，如先前报道的韦斯等人。在四周内传代4次后，通过每周更换培养基，使单个干细胞在不解离的情况下生长2个月。球体直径达到500-800 μ m，然后在4℃下在休眠培养基E（Gibco BRL）中保存一周。移植前即刻用PHK 26标记。在宿主成年大鼠中制备了两种不同类型的脑缺血。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-1，0.05ng/只）。移植冬眠的神经祖细胞球一周后证实（A）在MCA闭塞模型中缺血性病变的核心中没有移植物存活，（B）通过微弱的荧光标记物证实在缺血核心的边缘中的小簇存活，（C）在注射内皮素的纹状体中的大部分脑中的密集簇和迁移细胞。使用抗MAP-2和抗GFAP血清的免疫染色证明了移植物中的神经元和神经胶质细胞。在DMSO培养基中-180 ℃冷冻或在冬眠培养基中保存超过一周，都显示出鞭毛球，无法进行移植手术。本研究证明神经前体细胞球在保存一周后能够在脑内存活。少