【摘要】 【目的】探讨胚胎干细胞(ESCs)源性神经干细胞(NSCs)与海马源性NSCs的培养条件与分化特性,并进行比较研究。【方法】将拟胚体阶段视黄酸及视网膜Muler细胞诱导的ESCs和自60只BALB/c胎鼠海马组织分离的细胞(每次实验取10只),分别在NSCs选择性培养基中进行NSCs培养筛选,观察细胞形态变化,RT-PCR法检测Nestin基因表达,免疫细胞化学法检测Nestin等NSCs特异性标志物,对其分化后细胞检测Map2、Gap43、NF200、S100、GFAP神经组织细胞标志抗原,并对6次诱导结果进行观察比较。【结果】初级诱导的ESCs及海马细胞,在NSCs选择性培养基中培养,均形成大量呈Nestin抗原阳性,整合BrdU,表达Nestin基因的神经球样结构,且具有体外扩增传代及分化为神经元及神经胶质样细胞的能力。但ESCs源性NSCs增殖力更强,且分化细胞中NF200阳性细胞率42.1％±3.6％高于海马源性NSCs分化细胞中NF200阳性细胞率18.7％±5.1％,P<0.01。【结论】体外诱导可获得ESCs源性NSCs,与海马源性NSCs相比有更强的增殖力和向神经元分化的潜能,可作为干细胞移植治疗青光眼等神经变性疾病研究新的种子细胞。更多还原

【Abstract】 [Objective] To explore the culture condition and differentiation of embryonic stem cells(ESCs)-derived neural stem cells(NSCs) and hippocampus-derived NSCs in vitro. [Methods] Embryonic bodies induced by retinoic acid and retinal Miller cells were selected in NSCs-defined medium. So did cells isolated from hippocampus of 60 E18 BALB/c mice (10 mice in each experiment). The morphological changes and ability of expansion and differentiation of cells from two different origins were comparatively analyzed. Nestin antigen and BrdU incorporation were detected in the selected cells by immunocytochemistry and RT-PCR. [Results] Large amount of neuro-spheres were derived from both embryonic body and hippocampus in the selected medium, which could be passaged and differentiated, stained positive with Nestin and BrdU, and expressed Nestin gene. The percentage of NF positive cells obtained from differentiation of ESCs-derived NSCs was 42.1% ±3.6%, which was significantly higher than that from hippocampus-derived NSCs 18.7% ±5. 1%, P < 0.01. [Conclusion] NSCs derived from ESCs have potentially much more powerful ability than hippocampus-derived NSCs to expand and differentiate into neurons, and would play a new alternative role in stem cell transplantation to treat neuropathy of glaucoma and other retinal diseases.更多还原