The establishment of cell culture in hybrid type collagen and transplantation into intracerebral hemorrhage model rat brain

混合型胶原细胞培养的建立及脑出血模型大鼠脑移植

• 批准号: 10671300
• 负责人: KONDOH Takeshi
• 金额: $ 2.05万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671300/
• 关键词: Intracerebral hemorrhage; Neuronal Transplantation; Blood-brain Barrier; GFP; HUVEC; Electroporation; コラーゲン培地; 神経再生; 細胞移植

With a purpose of ex vivo gene transfer into injured brain such as intracerebral hemorrhage, following three sets of preliminary experiments were performed.(A) gene transfer by electroporation was attempted in the normal brain. The reporter gene pEGFP-Cl(25g /5 μl) 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 (200-400 mJ) 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.(B) Human umbilical vein endothelial cells (HUVECs) were transplanted in athymic mouse brain and neovascularization of grafled endothelial cells was studied. HUVECs were transfected by a reporter gene pEGFPE … More -N1 in vitro and grafted stereotactically in unilateral striatum of adult nude mice. Histological studies in four weeks revealed that grafted HUVECs newly formed microvessels in brain, which were migrated and fused with host vessels. Intravenous injection of Evans Blue prior to sacrificing animals resulted m 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 form a blood-brain barrier after grafting in the central nervous system.(C) 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 injecton (ET-1 ; 0.05 ng/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 to 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

为了将离体基因转移到损伤的脑例如脑出血中，进行了以下三组初步实验。(A)在正常脑中尝试通过电穿孔进行基因转移。将报告基因pEGFP-Cl（25 μ g/5 μl）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，高电压的脉冲引起广泛的组织损伤，而较低范围（200-400 mJ）的脉冲导致每个脑超过300个细胞的转染，这些细胞广泛分布在侧脑室的室管膜下区域，并将长突起延伸到纹状体中。(B)将人脐静脉内皮细胞（HUVECs）移植到裸鼠脑内，观察移植后内皮细胞的血管新生情况。用报告基因pEGFPE转染HUVECs ...更多信息 -N1在体外和立体定向移植在成年裸鼠的单侧纹状体。4周后的组织学观察显示，移植的HUVECs在脑内新生微血管，并与宿主血管融合。在处死动物之前静脉注射伊文思蓝没有导致染料外渗，表明移植的HUVEC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，来自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。(C)神经前体细胞在中枢神经系统损伤后的再生中已得到广泛的研究。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神经元和神经胶质细胞的移植。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

项目成果

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