不同体外诱导培养条件下新生鼠基底前脑神经干细胞增殖和分化为神经元的能力

目的：研究神经干细胞的增殖、迁移和分化可为揭示神经系统的发生、发育过程提供可靠依据。观察表皮生长因子和碱性成纤维生长因子在体外刺激新生鼠基底前脑神经干细胞的增殖情况，及其各自诱导神经干细胞分化成神经元的能力。 方法：实验于2005-12／2006—07在广州医学院解剖学教研室完成。①动物：清洁级新生24h内的SD大鼠30只，实验过程中对动物的处置符合动物伦理学标准。②实验方法：新生鼠在无菌条件下取脑，分离出基底前脑，胰蛋白酶消化，离心过滤制备单细胞悬液，接种于含B27的DMEM／F12培养基培养瓶中，每瓶40-60万个细胞，加入终浓度均为10μg／L的表皮生长因子和碱性成纤维生长因子刺激生长，在体外进行神经干细胞的克隆培养，传代培养过程中加入终浓度为6mg／LBrdU用于标记神经球，设立3组，各自加入体积分数为0．1的小牛血清、终浓度均为10μg／L的表皮生长因子、碱性成纤维生长因子，对培养得到的神经干细胞进行诱导分化。③实验评估：免疫荧光染色检测神经干细胞巢蛋白抗原的表达，并用BrdU标记和免疫荧光证实其增殖能力。免疫荧光染色检测不同诱导条件下神经干细胞向神经元分化的能力。 结果：①细胞形态观察：从新生鼠基底前脑成功分离出神经干细胞，原代培养呈透亮的圆球形，2—3d后细胞数目明显减少，部分细胞开始分裂。1周左右培养瓶中出现许多由数十到数百个细胞组成的悬浮生长的细胞球，球中的细胞形态规则，边界清楚，折光性较强，胞浆颜色较深，核，浆比较大。该细胞具有连续增殖能力，可以传代培养。②巢蛋白抗原的表达：传代神经球中的细胞均呈巢蛋白抗原阳性。③BrdU标记检测：克隆球中的细胞均为BrdU阳性，表明克隆球是由不断分裂增殖的细胞组成。④诱导分化结果：?

Proliferation and differentiation of neural stem cells from neonatal rat basal forebrain of newborn rats into neurons in different culture conditions

AIM： To study the proliferation, migration and differentiation of neural stem cells to give a useful proof of appearance and development of nervous system, and to observe the effects of epidermal growth factor （EGF） and basic fibroblast growth factor （bFGF） on the proliferation and the ability of differentiation into neurons of neural stem cells-derived from basal forebrain in vitro.
METHODS： Experiments were performed at the Department of Anatomy, Guangzhou Medical College from December 2005 to July 2006. ① Thirty SPF class new-born SD rats （less than 24 h） were selected. All the manipulation was according to animal ethnic standards. ②The brains of new-born rats were taken out under aseptic condition. The single cell suspension from the brain tissues of the basal forebrain by gentle mechanical dissociation with the use of trypsin was cultured in DMEM/F12 medium containing B27, EGF and bFGF （10 μg/L）, （4-6）× 10^5 cells/flask. Neurospheres were generated and floated in the culture. Neurospheres were collected and labeled with BrdU （6 mg/L） in the culture medium. The experiments were divided into three groups： adding 0. 1 （V/V） bovine serum, EGF （10 μg/L） or bFGF （10 μg/L） into the culture. ③BrdU labeling was used to confirm the proliferation potential. The expression of Nestin antigen, BrdU labeling and the ability of differentiation into neurons of neural stem cells were detected by immunofluorescence techques.
RESULTS： ①Morphology observation： Neural stem cells-derived from basal forebrain of new-born rats were bright ball shape in primary culture. The number of cells decreased after primary culture and some cells began to divide. One week after culture, the neurospheres were made of decades and hundreds of cells. The shape and verge of neural stem cells was regular and clear, and refraction ability was strong. The color of cytoplasm was deep and ratio of nucleus/cytoplasm was big. These results showed neural stem cells-derived from basal forebrain ha