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

在本研究中，我们首次研究了人类畸胎瘤细胞系（NT-2）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维甲酸对体外培养的畸胎瘤细胞（NT-2）处理5周，成功获得神经样细胞（NT-2N）。然后研究NT-2N细胞和NT-2细胞作为神经元细胞的电生理特性。我们发现在NT-2N细胞中观察到电压敏感的Na+电流，而在NT-2细胞中则没有，并且除了谷氨酸受体外，NT-2N细胞还表达GABA-A受体，这表明这些受体在大脑发育早期的神经传递中起着至关重要的作用。其次，从大鼠胎儿纹状体中分离神经干细胞，在含血清无表皮生长因子的培养基中培养2个月，不分离。当在含血清的培养基中复制时，每个未传代的s . More细胞保持分化为神经元和神经胶质细胞的潜力。电镜观察显示细胞未成熟，胞质较暗或处于增殖阶段。细胞在球内分化不均；有的具有粗糙的内质网，有的具有长突，中间有丝。在球内也观察到细胞凋亡。这些在球的核心中占主导地位，在大的细胞外空间中观察到大量的细胞碎片，包括凋亡小体。这些具有持续分化细胞的神经球可以作为神经元移植的替代来源。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产生人类星形胶质细胞细胞系。将这些星形胶质细胞冷冻保存后，经环孢素免疫抑制后移植到正常大鼠纹状体中。人特异性胶质纤维酸性蛋白抗原免疫组化研究显示，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内。移植物内存在大鼠特异性内皮屏障抗原（EBA）阳性的血管，表明移植物的血管生成是由宿主来源的内皮细胞完成的。EBA的表达也提示异种星形胶质细胞移植可能形成血脑屏障。在移植后2周停止免疫抑制的动物中，移植物能够再存活3周，然而，在停止免疫抑制后8周出现排斥反应。这表明屏障作为一种免疫特权的形成是不完整的。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具有显著的融合性，星形胶质细胞自体移植可能具有治疗目的。少

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