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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

在正常脑内尝试电穿孔基因转移。将报告基因pEGFP-C1（25g/5μl）注射到幼龄成年大鼠纹状体中，并通过放置在纹状体中的一对电极施加不同范围的方形电脉冲。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（200-400mJ）导致每脑300多个细胞转染，这些细胞广泛分布在侧脑室室管膜下区，并延伸到纹状体。将人脐静脉内皮细胞（HUVECs）移植到胸腺发育不全的小鼠脑内，观察其新生血管的形成。用报告基因pEGFPE-N1转染huvea体外，立体定向移植到成年裸鼠单侧纹状体。4周的组织学研究表明，移植的HUVECs在脑内新形成了更多的微血管，并与宿主血管发生了迁移和融合。在献祭动物之前静脉注射Evans Blue没有导致染料外溢，这表明移植的HUVECs形成了血脑屏障。免疫组织化学表明，宿主星形胶质细胞在移植的内皮细胞上延伸胶质足，部分新形成的血管中葡萄糖转运蛋白-1阳性。这些结果表明，来自异位来源的内皮细胞在中枢神经系统移植后具有脱离血脑屏障的潜力。神经祖细胞在损伤中枢神经系统再生中的应用已得到广泛的研究。对于这些细胞的移植，有报道表明，移植后存活率提高的不是单纯的单细胞悬浮液，而是由祖细胞组成的球体。在本研究中，从E-14大鼠脑室下区获得大鼠神经干细胞，然后在含有egf的培养基中自由漂浮培养，如Weiss等人先前报道的那样。在四周内传代四次后，通过每周更换培养基，使单个干细胞在没有分离的情况下生长两个月。球体直径500 ~ 800um，在冬眠培养基E （Gibco BRL）中4℃保存一周。接枝前用PHK26标记球。制备了两种不同类型的成年大鼠脑缺血模型。一种是光化学法封堵大脑中动脉，另一种是单侧纹状体内注射内皮素（ET-1, 0.05ng/只）。冬眠神经祖细胞球移植1周后，发现(A) MCA闭塞模型缺血病灶中心无移植物存活，(B)缺血病灶边缘荧光标记显示小簇细胞存活，(C)注射内皮素纹状体中大部分脑内密集簇细胞和迁移细胞存活。抗map -2和抗gfap血清免疫染色显示移植物中有神经元细胞和胶质细胞。在含dmso的培养基中-180℃低温保存或在冬眠培养基中保存超过一周，显示出易脱落的球体，无法处理移植手术。本研究表明，神经元祖细胞球在大脑中保存一周后仍能存活。少