The basic research for the establishment of the donor for the neural transplantation with the miniature swine neuroepitherial stem cells.

小型猪神经上皮干细胞神经移植供体建立的基础研究。

基本信息

批准号：
14571331
负责人：
UCHIDA Koichi
金额：
$ 1.6万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571331/
关键词：
neural transplantation neuroepithelial stem cells xenograft functional effect donor miniature swine synapse neural circuitry reconstruction neuroepithelial stem cells neuroepithelial stem cell mesencephalic neural plate xenografting

项目摘要

The tissue and cell dissociation culture of the embryonic day 10.5 (E10.5) Wister rats mesencephalic neural plate, consisted wife neuroepitheral stem cells (NESCs). were cultured in vitro under various conditions and were analyzed 7 days after the primary culture. When they were cultured as a tissue, almost all cells differentiated into neurofilament (NF) positive neuron extending long neurites in a serum-free and serum containing medium. But, there were no astrocyte expressed glial fibrially acidic protein (GFAP). For the evaluation of the naerochemical marker, many glutamate positive cells, few γ-aminoburyric acid (GABA) and tyrosine bydroxylase (TH) positive cells were there. On the other hand, when they were plated in a dissociation culture, neurosphere generated 7 days after the primary cell culture in the medium containing fibroblast growth factor-2 (FGF-2). The FGF-2 responsive neurosphere were positively stained by nestin and able to maintain. Under the condition of differentia … More tion all cells of the primary neurosphere differentiated into NF positive neuron, not into GFAP positive cell, in the differentiated neurosphere, many glutamate positive cells few GABA and TH positive cells were there as same as the tissue culture. These indicated that the rat mesencephalic neural plate cells can keep the neuronal differentiation tendency until one week by FGF-2 administration.We also analyzed immunohisochemically the biological nature of the mesensephalic neural plate and FGF-2 responsive neurospheres transplanted into the normal adult rat brain. The viable graft in the host brain was observed for several weeks after transplantation and many Hu-positive neuronal cells and a few GFAP-positive cells were seen in the graft Especially in the FGF-2 responsive graft marry. Hu-positive neuronal cells and a few nestin-positive cells were observed, but no GFAP-positive cells could be identified 2 weeks after transplantation. These results suggested that NESCs demonstrated a propensity to differentiate into neurons in the adult brain environment.The mesencephalic neural tube tissue graft from E17-18 swine embryos implanted immediately into the strianum of the Parkinsonian model rat were observed for several weeks with 1-week administration of the immunosuppressants after transplantation only. The swine derived grafts had many NF-positive and TH-positive cells in the host rat striatum. An electron microscopic study revealed both efferent and afferent synaptic formations in the donor-derived immature neurons or TH-positive donor cells in the grafts. Myelimated axons, bath positive and negative for swine-specific NF antibody, were mingled together in the graft.Both mini-swine derived NESCs and rat derived NESCs survived in Parkinsonian rat striatum at 8 weeks after transplantation. The rat NESCs grafted rat showed complete recovery from Parkinsonian behavioral defects in amphetamine-induced rotation test. On the other hand, the mean number of amphetamine-induced rotations made by the mini-swine NESCs grafted rats expressed as a percentage of the number before grafting had not changed until 4 weeks after grafting but decreased to 30% at 8 weeks after grafted in the grafts of both mini-swine NESCs and rat NESCs, many TH positive donor-derived cells were there 2 weeks after transplantation for the immunohistolgical evaluation.NESCs are therefore an attractive candidate as a source of donor material for intracerebral grafting in neural circuitry reconstruction Less

胚胎第10.5天（E10.5）的组织和细胞解离培养物，wister大鼠messencephalic neuroplate，是妻子神经雌性干细胞（NESC）。在各种条件下在体外进行培养，并在初级培养后7天进行分析。当它们作为组织培养时，几乎所有细胞分化为神经丝（NF）阳性神经元，在无血清和含有培养基的血清中延伸长神经元。但是，没有星形胶质细胞表达神经胶质的纤毛酸性蛋白（GFAP）。为了评估Naerochemical标记物，许多谷氨酸阳性细胞，很少有γ-氨基苯甲酸（GABA）和酪氨酸抑制酶（Th）阳性细胞在那里。另一方面，当它们被镀在分离培养物中时，神经球在原代细胞培养后7天产生，其中含有成纤维细胞生长因子-2（FGF-2）。 FGF-2反应性神经球被巢蛋白阳性染色并能够维持。在差异的条件下……更多的原代神经圈的细胞分化为NF阳性神经元，而不是GFAP阳性细胞，在分化的神经圈中，许多谷氨酸阳性细胞很少有GABA和Th阳性细胞与组织培养一样。这些表明，大鼠膜脑神经填充细胞可以保持神经元分化趋势，直到通过FGF-2给药一周才能保持神经元的分化趋势。我们还分析了免疫异性发育的生物学性质，中素神经镀和FGF-2响应性神经圈的生物学性质被移植到正常的成年大鼠大脑中。移植后几周观察到宿主大脑中的活粒，许多HU阳性神经元细胞和几个GFAP阳性细胞在移植物中尤其是在FGF-2反应式谷物结婚中。观察到神经元细胞和一些Nestin阳性细胞，但是在移植后2周无法鉴定GFAP阳性细胞。这些结果表明，NESC证明了在成人大脑环境中分化为神经元的希望。从E17-18猪胚胎中，立即植入了帕金森尼模型大鼠的Strianum的Messencephalic神经元管组织移植物，仅通过1周的施用了1周的免疫抑制剂。猪衍生的图形在宿主大鼠纹状体中具有许多NF阳性和TH阳性细胞。一项电子显微镜研究揭示了供体衍生的未成熟神经元或移植物中的Th阳性供体细胞中有效和传入的突触地层。在移植后8周，在帕金森氏大鼠纹状体中，骨髓特异性的NF抗体的骨髓轴突，淋浴和阴性的NF抗体的阳性和阴性。大鼠NESC接枝大鼠在苯丙胺诱导的旋转试验中完全从帕金森氏症行为缺陷中恢复了完全恢复。 On the other hand, the mean number of amphetamine-induced rotations made by the mini-swine NESCs grafted rats expressed as a percentage of the number before grafting had not changed until 4 weeks after grafting but decreased to 30% at 8 weeks After grafted in the grafts of both mini-swine NESCs and rat NESCs, many TH positive donor-derived cells were there 2 weeks after transplantation for the因此，免疫史学评估。因此，NESCS是一种有吸引力的候选者，作为供体材料的来源