维甲酸诱导胚胎大鼠脑海马神经干细胞向神经元的分化

Retinoic acid induces the differentiation of neural stem cells into neurons in the embryonic hippocampus of rats

背景:目前认为直接进行神经干细胞移植后细胞虽能存活,但是只分化成神经胶质细胞,并不能分化成有功能的神经元。目的:探讨维甲酸诱导对胚胎大鼠脑海马神经干细胞向神经元分化的作用。设计、时间及地点:细胞学体外实验,于2008-09/2009-02在辽宁医学院科技实验楼完成。材料:胚龄13.5d的SD大鼠由辽宁医学院实验动物中心提供。方法:分离胎鼠脑海马组织,胰蛋白酶消化法体外培养获得神经干细胞。将原代和传代细胞以1×107L-1接种到培养孔中,分别进行常规贴壁分化培养和维甲酸诱导分化培养。主要观察指标:神经干细胞的鉴定,光镜及免疫组化检测神经干细胞诱导分化结果。结果:免疫组织化学检测结果显示,原代和传代后得到的神经干细胞团均呈巢蛋白阳性。常规贴壁分化培养7d后,神经元多呈椭圆型和近似三角形,胞体大,细胞边缘清楚,胞体上有多个突起;而维甲酸诱导分化培养后,神经元数量增加,形态清楚,但细胞胞体上突起较少。与常规贴壁分化培养比较,维甲酸诱导分化培养后神经干细胞向神经元分化率明显升高(P<0.01),神经干细胞向神经胶质细胞分化率明显降低(P<0.01)。结论:从大鼠胚胎脑海马组织中分离得到可自我复制和多向分化的神经干细胞,维甲酸体外诱导后可以增加其向神经元方向分化的比例。

BACKGROUND：Cells can survive following direct neural stem cell transplantation,but only can differentiate into glial cells,other than neurons. OBJECTIVE：To explore retinoic acid induction effects on differentiation of neural stem cells into neurons in the hippocampus of embryonic rats. DESIGN,TIME AND SETTING：The cytological in vitro study was performed at the Technological Experiment Building of Liaoning Medical University from September 2008 to February 2009. MATERIALS：Sprague Dawley rats at embryonic age of 13.5 days were supplied by Experimental Animal Center,Liaoning Medical University. METHODS：The tissue mass of hippocampus from embryonic day 13.5 rats was isolated under the anatomical microscope. The mass was digested with trypsin to obtain neural stem cells. Primary cultured and passaged cells were cultured in a plate at 1×107/L for conventional adherence culture and retinoic acid induction culture. MAIN OUTCOME MEASURES：Induction and differentiation of neural stem cells were identified by immunohistochemistry under an optical microscope. RESULTS：The results of immunohistochemistry detection showed that neural stem cells were positive for Nestin. At 7 days following conventional culture,neurons were ellipse and subtriangular,with big body and clear boundary and many processes. Following retinoic acid culture,the number of neurons was increased,with clear morphology and a few processes. Compared with the conventional culture,the differentiation rate from neural stem cells into neurons was significantly greater following retinoic acid culture（P 0.01） ,and the differentiation rate from neural stem cells into glial cells was significantly reduced（P 0.01） . CONCLUSION：The embryonic hippocampus of rats contained the self-production and multi-directional differentiation of neural stem cells. Retinoic acid can drive the proportion of neural stem cells into neurons.