胚胎大鼠原代神经干细胞培养方法的改进

背景:神经干细胞增殖分化受体内外微环境的影响,不同细胞培养液对细胞的增殖分化有不同的作用.目的:改进传统上应用无血清培养液培养原代神经干细胞的方法,寻找一种快速便捷的培养方式.设计、时间和地点:随机对照细胞实验,于2005-11/2006-09在青岛大学医学院脑科研究所完成.材料:选用孕后12～16 d 的Wistar大鼠10只,取胎鼠脑组织制成细胞悬液.方法:将配置的细胞密度约1×109个/mL滤液接种入4个50 mL细胞培养瓶,分为2组:对照组和实验组,每组2个培养瓶.对照组加入DMEM/F12、2?7型人类白细胞抗原、20 mg/L的碱性成纤维细胞生长因子,20 mg/L的表皮生长因子无血清培养液,实验组加入DMEM/F12、5%胎牛血清,两三天后换为与对照组相同的无血清培养液.主要观察指标:应用倒置显微镜和免疫细胞化学法观察两组神经干细胞的增殖生长状况.结果:神经干细胞生长状态:①两组大多数细胞表达神经干细胞的特征性标志抗原神经巢蛋白,免疫染色呈阳性反应.②实验组神经干细胞的聚球速度明显快于对照组,可提前三四天观察到神经干细胞球.③两组细胞数量基本无差异,实验组细胞球体较对照组大.经血清培养液诱导分化后部分呈神经元特异性烯醇酶阳性,部分呈神经胶质酸性蛋白阳性,表明神经干细胞已分化为神经元样细胞和神经胶质细胞.结论:含血清培养液预先培养可以加快神经干细胞增殖生长速度.

A new method to culture primary neural stem cells of embryonic rats

BACKGROUND:Both in vivo and vitro microenvironment can influence the proliferation and differentiation of neural stem cells (NSCs). In addition, cell culture solution plays variable roles in cell proliferation and differentiation.OBJECTIVE:To develop a convenient and rapid method to promote NSC primary culture by modifying traditional serum-free medium. DESIGN, TIME AND SETTING:Randomized controlled cell trial was performed at Institute of Brain Science, Qingdao University Medical School from November 2005 to September 2006.MATERIALS:Ten Wistar rat of gestation for 12-16 days; cell suspension fron brain tissue of embryonic rat. METHODS:Cell suspension were seeded into four 50-mL culture flasks with cell density of 1×106 mL-1, and divided into 2 groups with 2 flasks in each group. The control cells (flasks A and B) were cultured in serum-free medium containing DMEM/F12, B27 (2%), basic fibroblast growth factor (20 μg/L), and epidermal growth factor (20μg/L), and the experimental cells (flasks C and D) were firstly cultured with DMEM/F12 containing fetal bovine serum (FBS, 5%), following by the same serum-free medium 2 to 3 days later. MAIN OUTCOME MEASURES:The proliferation of NSCs was observed by inverted microscopy and immunocytochemistry.RESULTS:①Most of the cells in two culture conditions expressed nestin, the specific marker of NSCs, and were immunocytochemically positive. ②Neural stem cells cultured with FBS formed neurospheres 3-4 days earlier than those without FBS. ③There were no significant differences in cell number, but the neurospheres under the experimental condition were larger than those in control group. Some of the cells were neurone specific enolase-positive after serum-conditioned induction, and some were glial fibrillary acidic protein-positive, indicating NSCs had differentiated into neuron-like cells and neurogliocytes.CONCLUSION:FBS-conditioned pre-culture can accelerate the proliferation of neural stem cells.