胚胎大鼠脊髓神经干细胞体外培养与定向分化为胆碱能神经元的研究

THE CULTURE AND SPECIFIC DIFFERENTIATION INTO CHOLINERGIC NEURONS OF EMBRYONIC SPINAL CORD STEM CELLS OF RAT IN VITRO

为了研究胚胎大鼠脊髓源神经干细胞(ESCSCs)的培养和体外分化为胆碱能神经元的情况,本文从孕龄13d的胚胎大鼠脊髓组织中分离获得神经干细胞,采用含表皮生长因子(EGF)及碱性成纤维细胞生长因子(bFGF)的无血清限定性培养基培养,并通过添加维甲酸(RA)、音猬因子(SHH)和神经营养因子-3(NT-3)等诱导因子,进行干细胞体外诱导,用免疫荧光细胞化学方法观测其向胆碱能神经元分化的情况。结果表明:从胚胎脊髓中可分离得到大量的神经干细胞,通过含EGF和bFGF的限定性培养基培养可获得干细胞球,通过添加RA、SHH和NT-3等诱导因子后,可见有胆碱能神经元生成。本研究结果提示,胚胎大鼠脊髓神经干细胞在添加EGF与bFGF的限定性培养基中可以增殖并长期保持稳定的ESCSCs性状,在添加特殊因子的条件下,并在体外ESCSCs可以被定向诱导分化为胆碱能神经元。

To study the cultivation and the specific differentiation of embryonic spinal cord stem cells （ESCSCs）, the neural stem cells from spinal cord of 13 d fetal rats were harvested and cultivated in a serum-free limited medium which contains epidermal growth factor （EGF） and basic fibroblast growth factor （ bFGF）, and the differentiation of these cells into cholinergic neurons was observed by immunocytochemistry after exposure of the cells to retinoic acid （ RA）, sonic hedgehog （ SHH ）, neurotrophin-3 （ NT-3 ）. The results indicate that lots of neural stem cells can be obtained from the embryonic spinal cord, and neurospheres could be formed through cultivation in limited medium containing EGF and bFGF. In addition, after exposed in specific factors such as RA, SHH, NT- 3, the stem cells could be induced to differentiate into cholinergic neurons, These data suggest that the limited medium containing EGF and bFGF used in the present study can increase the proliferation and maintain the attributes of ESCSCs in vitro for a prolonged period, and most importantly, ESCSCs can be directly induced to differentiate into cholinergic neurons in vitro after exposure in specific factor.