神经干细胞培养及其影响因素

背景：神经干细胞移植治疗是中枢神经系统损伤性、难治性疾病研究的热点。如何有效提高神经干细胞存活率、克隆形成率将是其应用的重要基础和前提。目的：探讨影响神经干细胞培养质量的可能因素,为神经干细胞的基础和临床应用研究提供参考。方法：①神经干细胞分离和培养：分离出生24h内SD大鼠的大脑,剪碎离心后以1×108L-1的浓度接种,经含体积分数为10%胎牛血清的DMEM/F12培养基预培养4h后改用无血清条件培养基（DMEM/F12,碱性成纤维细胞生长因子、表皮生长因子、B27）培养,2d或3d换液1次,6d时传代。②神经干细胞鉴定：倒置显微镜下观察细胞形态及生物学特性;免疫组化检测第3代细胞神经巢蛋白表达和诱导分化后神经元特异性烯醇化酶和神经胶质酸性蛋白的表达。结果与结论：出生24h的新生大鼠可获得足量的神经干细胞;血清预培养可提高神经干细胞的增殖和存活率;免疫组化结合血清诱导分化可对培养的神经干细胞进行定性鉴定。取材时间、细胞接种密度、传代时间、细胞因子、血清等多种因素都将影响神经干细胞的培养质量。

Culture of neural stem cells and the influencing factors

BACKGROUND： Neural stem cells transplantation is the research hotspot for the treatment of central nervous system damage and refractory disease. How to improve the survival rate and the cloning formation rate of neural stem cells will be an important foundation and prerequisite for its application. OBJECTIVE： To study the influencing factors on the culture of neural stem cells in vitro in order to provide reference for the basic and clinical application of neural stem cells. METHODS： Neural stem cells were isolated from the brain of Sprague-Dawley rats within 24 hours afterbirth, centrifuged, and then inoculated at the concentration of 1×108 /L. Then the cells were pre-cultured with dulbecco’s modified eagle’s medium/F12 medium containing 10% fetal bovine serum for 4 hours and then serum-free conditioned medium （dulbecco’s modified eagle’s medium /F12, basic fibroblast growth factor, epidermal growth factor and B27） was used. The medium was changed every 2 or 3 days, and cells were passaged at 6 days. Morphological and biological characteristics of the cells were observed under inverted microscope to indentify neural stem cells. Immunocytochemistry was used to examine nestin expression on passage 3 neural stem cells and the expression of neuron-specific enolase and glial fibrillary acidic protein in differentiated cells. RESULTS AND CONCLUSION： Neural stem cells could be obtained from newborn rats 24 hours after birth. Pre-culture with serum could increase the proliferation rate and survival rate. Immunohistochemistry combined with serum-induced differentiation could be used to identify neural stem cells. Sampling time, cell density, passage time, cytokines and serum all can influence the proliferation and differentiation of neural stem cells.