"The roles of neurotrophic factors and nitric oxide on the differentiation of human neuronal cells"

“神经营养因子和一氧化氮对人类神经元细胞分化的作用”

• 批准号: 08671584
• 负责人: KONDOH Takeshi
• 金额: $ 0.58万
• 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: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671584/
• 关键词: Neuronal Stem Cell; Teratocarcinoma; Retinoic Acid; Transplantation; Astrocytes; GABA receptor; Apoptosis; Blood-Brain-Barrier; Cryopreservation; retinoic acid; whole-cell patch-clamp; アセチルコリン受容体; 神経細胞移植; NADPH-d; NT-2

In the present study, we have first examined the possibility of neuronal differentiation of human teratocartinoma cell line (NT-2) with a purpose of alternative source for neuronal transplantation. By means of treating the teratocarcinoma cells (NT-2) in vitro with 10 mM of retinoic acid for five weeks, neuronal like cells in morphology (NT-2N) were successfully obtained. NT-2N cells as well as NT-2 cells were then studied electrophysiologicaly for their property as neuronal cells. We have found that voltage sensitive Na+ currents were observed in NT-2N cells but not in the NT-2 cells and that in addition to the glutamate receptors, the NT-2N cells expressed GABA-A receptors, suggesting that those receptors have a crucial role in neurotansmission from the earlier stage of the brain development.Second, neural stem cells isolated from rat fetus striatum were cultured in serum free epidermal growth factor containing medium as spheres for two months without dissociation. Each nonpassaged s … More phere maintained the potential to differentiate to neurons and glia cells when replated in serum containing medium. The electronmicroscopical study revealed immature cells with scant dark cytoplasm or in proliferation stage. The cells differentiated heterogeneously in the sphere ; some had rough endoplasmic reticula and others had long processes with intermediate filaments. Cells undergoing apoptosis were also observed in the sphere. Those were dominant in the core of spheres where abundant cellular debris including apoptotic bodies were observed in the large extracellular spaces. These neural spheres with ongoing differentiation of the cells could fit as an alternative source for neuonal transplantation.Third, a human astrocyte cell line was generated from a tissue of white matter containing reactive astrocytes in a routine neurosurgical procedure. Those astrocytes were cryopreserved and grafted into normal rat striatum with immunosurpression by cycrosporine. Immunohistochemical study with human specific glial fibrillary acidic protein antigen revealed that grafted astrocytes formed dense cell clusters in the host brain and that part of the cells migrated away into the host striatum. Inside the grafts, there were vessels which were positive with rat specific endothelial barrier antigen (EBA), suggesting that the angiogenesis of the grafts was performed by the host-derived endothelial cells. The expression of EBA also suggests that the blood-brain-barrier was likely formed in the xenograft of astrocytes. In the animals in which the immunosurpression discontinued at two weeks postgrafting, the grafts were able to survive for three additional weeks, however, rejected at eight weeks after discontinuation of the immunosurpression. This indicated that the barrier formation as an immunological privilege was incomplete. Our study indicates that although the formation of blood-brain-barrier is incomplete in the xenografts of astrocytes, the grafted astrocytes themselves were remarkablely integrated with host circumstances and that the autograft of the astrocytes might be feasible for the therapeutic purpose. Less

在本研究中，我们首先研究了人类畸胎瘤细胞系(NT-2)向神经元分化的可能性，目的是为神经元移植提供替代来源。用10 mM维甲酸处理体外培养的畸胎癌细胞(NT-2)5周，成功地获得了形态类似神经元样细胞(NT-2N)。然后对NT-2N细胞和NT-2细胞作为神经细胞的特性进行了电生理学研究。我们发现在NT-2N细胞中有电压敏感的Na+电流，而在NT-2细胞中则没有。除了谷氨酸受体外，NT-2N细胞还表达GABA-A受体，这表明这些受体在脑发育早期的神经传递中起着至关重要的作用。第二，从大鼠胚胎纹状体分离的神经干细胞在含血清表皮生长因子的培养液中培养两个月，不分离。每个未通过的S…当在含血清的培养液中复制时，More phere保持了向神经元和神经胶质细胞分化的潜力。光镜下可见未成熟细胞胞质稀少，深色或处于增殖期。细胞在球体内异质分化，有的有粗面内质网，有的有长突起，中间有细丝。球体内也可观察到正在发生凋亡的细胞。这些细胞主要分布在球体的核心，在较大的细胞外间隙中观察到大量的细胞碎片，包括凋亡小体。这些不断分化的神经球可以作为中性移植的替代来源。第三，在常规神经外科手术中，从含有反应性星形胶质细胞的白质组织中培养出人类星形胶质细胞系。将这些星形胶质细胞冷冻保存后移植到正常大鼠纹状体，并用环孢素进行免疫抑制。用人类特定的胶质纤维酸性蛋白抗原进行的免疫组织化学研究表明，移植的星形胶质细胞在宿主脑内形成致密的细胞团，部分细胞迁移到宿主纹状体。移植物内可见大鼠特异性内皮屏障抗原(EBA)阳性的血管，提示移植物的血管生成是由宿主来源的内皮细胞完成的。EBA的表达也提示血脑屏障可能是在星形胶质细胞异种移植中形成的。在移植后两周停止免疫抑制的动物中，移植物能够额外存活三周，但在停止免疫抑制后八周被拒绝。这表明作为免疫豁免的屏障的形成是不完整的。我们的研究表明，尽管星形胶质细胞异种移植的血脑屏障形成不完全，但移植的星形胶质细胞本身与宿主环境有显著的结合，星形胶质细胞自体移植可能是可行的治疗目的。较少

项目成果

T.Kondoh, T.Nishizaki, N.Tamaki, T.Matsuoka, H.Akiyama, M.Mori, Y.Okada: "Expression of neurotransmitter receptor and grafting of human neurons derived from NT-2 cells." Soc Neurosci Abstr. 23 Part1. 345 (1997)

T.Kondoh、T.Nishizaki、N.Tamaki、T.Matsuoka、H.Akiyama、M.Mori、Y.Okada：“神经递质受体的表达和源自 NT-2 细胞的人类神经元的移植。”

秋山英之: "ヒトアストロサイトのラット脳内移植后の組織像" 神経組織の成長・再生・移植. 9. 61-62 (1997)

Hideyuki Akiyama：“将人星形胶质细胞移植到大鼠大脑后的组织学图像”神经组织的生长、再生和移植（1997）。

Matsuoka Toshiyuki, Takeshi Kondoh, Norihiko Tamaki, Tomoyuki Nishizaki: "The GABA-A receptor is expressed in human neurons derived from a teratocarcinoma cell line." Biochemical and Biophysical Research Communication. 237. 719-723 (1997)

Matsuoka Toshiyuki、Takeshi Kondoh、Norihiko Tamaki、Tomoyuki Nishizaki：“GABA-A 受体在源自畸胎癌细胞系的人类神经元中表达。”

T.Kondoh, E.Fujimoto, H.Akiyama, N.Tamaki: "Prolireration and differentiation of long-term cultured nonpassage neural spheres" The Abstracts for the Japanese Society for Neural Growth, Regeneration and Transplantation. 9. 97-98 (1997)

T.Kondoh、E.Fujimoto、H.Akiyama、N.Tamaki：“长期培养的非通道神经球的增殖和分化”日本神经生长、再生和移植学会摘要。

N.Nishizaki, T Kondoh, T.Matsuoka, N.Tamaki: "GABA-A receptor is expressed in human neurons derived from a teratocarcinoma cell line (NT2)." The Abstracts for the Japanese Society for Neural Growth, Regeneration and Transplantation. 9. 71-72 (1997)

N.Nishizaki、T Kondoh、T.Matsuoka、N.Tamaki：“GABA-A 受体在源自畸胎癌细胞系 (NT2) 的人类神经元中表达。”