课题背景：课题获得广东省自然科学基金项目资助，目前取得的研究成果包括：①成功从基底前脑培养获得神经干细胞，在体外诱导条件下可使神经干细胞分化为神经元的比例显著增加。②成功制备了神经生长因子缓释微球，微球所载的神经生长因子在体外或脑内均能够持续释放４周以上。③神经生长因子缓释微球移植入基底前脑，能够保护该区的胆碱能神经元免受隔－海马伞引起的逆行性溃变。

应用要点：①表皮生长因子和碱性成纤维生长因子作为神经干细胞增殖的刺激剂长期被人们应用，但是它们各自对神经干细胞分化的影响报道甚少。②通过多重荧光标记技术，能够更直观地显示实验结果。

偏倚或不足：：①实验所用神经干细胞是从原代取材，经传代培养获得，纯度很可能低于神经干细胞系。②可以设计一组从其他部位取材所得的神经干细胞进行诱导分化作为对照研究，这样可更全面地说明不同体外诱导培养条件下对神经干细胞增殖和分化为神经元能力的影响。③若能够做到单细胞克隆，试验结果的可控性会更高一些。

广州医学院，1组织胚胎学教研室，2解剖学教研室， 3药理学教研室，广东省广州市
510182

谷海刚☆，男，1976年生，河南省伊川县人，汉族，北京协和医学院(清华大学医学部)毕业，博士，讲师，主要从事神经系统疾病干细胞治疗和新型给药系统方面的研究。guhaigang@yahoo.
com.cn

通讯作者：龙大宏，博士，教授，广州医学院解剖学教研室，广东省广州市 510182
dahonglong88@ yahoo.com.cn

广东省自然科学基金(06022674) *

摘要
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24 h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6 mg/L BrdU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，2~3 d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：在体积分数为0.1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诱导条件下，神经干细胞分化为神经元的比例分别为20%，22%，40%。
结论：①表皮生长因子和碱性成纤维生长因子在体外能够刺激新生鼠基底前脑神经干细胞连续增殖，且具有胚胎源性。②以血清作为对照，碱性成纤维生长因子诱导神经干细胞向神经元分化的能力强于表皮生长因子。
关键词：神经干细胞；神经元；基底前脑；表皮生长因子；碱性成纤维生长因子

谷海刚，龙大宏，李晓滨，张贵平，罗媚，李佳楣，冷水龙. 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[J].中国组织工程研究与临床康复，2008，12(8):1445-1448
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1445(ps).pdf]

中图分类号:R394.2
文献标识码:B
文章编号:1673-8225
(2008)08-01445-04

收稿日期：2007-09-14
修回日期：2007-12-05
(07-50-9-5050/ZS·Q)

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

Abstract

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)×105 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 had the ability of self-renewing and could be obtained by subculture. ②Nestin expression: All the generated cells showed Nestive positive. ③BrdU labeling detection: All the neurospheres were BrdU positive, showing neurospheres were made of sustained self-renewing cells. ④Differentiation results: Under the conditions of 0.1 (v/v) bovine serum, EGF (10 μg/L) and bFGF (10 μg/L) into the culture, the ratios of neural stem cells differentiating into neurons were 20%, 22% and 40%, respectively.
CONCLUSION：① EGF and bFGF can activate neural stem cells-derived from basal forebrain of new-born rats to proliferate and make them maintain embryo characteristics. ② Serum serving as a control group, the ability of bFGF differentiating neural stem cells into neurons is stronger than that of EGF.

Gu HG, Long DH, Li XB, Zhang GP, Luo M, Li JM, Leng SL. Proliferation and differentiation of neural stem cells from neonatal rat basal forebrain of newborn rats into neurons in different culture conditions.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(8):1445-1448(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1445(ps).pdf]

0 引言

1992年，Reynolds等[1]从成年小鼠纹状体中分离培养出一群细胞，这些细胞可以在体外自我增殖，并能够分化为神经元和胶质细胞，后被定义为神经干细胞。神经干细胞的体外培养、定向诱导分化的神经生物学研究及其在临床上的应用已成为21世纪神经科学的一个研究热点[2-4]。利用培养的神经干细胞所进行的研究明显促进了对神经发育机制的了解，一些能够调节神经干细胞增殖、分化的细胞因子将直接应用于控制体内细胞的增殖和分化，为某些神经系统疾病如神经系统退变性疾病、神经系统发育异常的治疗开拓了新的前景[5-8]。现在多数脑区都存在而且培养得到了神经干细胞，基底前脑也存在神经干细胞，不同条件对基底前脑神经干细胞分化的影响还未见报道。

1 材料和方法

设计：细胞观察实验。
单位：广州医学院组织胚胎学教研室，解剖学教研室，药理学教研室。
材料：实验于2005-12/2006-07在广州医学院解剖学教研室完成。清洁级新生24 h内的SD大鼠30只（由中山大学实验动物中心提供，动物质量合格证号：0008780），体质量15~25 g，雌雄不拘，实验过程中对动物的处置符合动物伦理学标准。DMEM/F12（1∶1）基础培养基，B27无血清添加剂（GIBCO）；表皮生长因子，碱性成纤维生长因子（GIBCO）；50 mL培养瓶（CORNING）；24孔塑料培养板（NUNC）；小鼠抗巢蛋白单克隆抗体，兔抗神经微丝多克隆抗体(Chemicon)；小鼠抗BrdU单克隆抗体，羊抗小鼠IgG-FITC，BrdU（Sigma）；羊抗兔IgG-cy3 （Chemicon）。
设计、实施、评估者：设计为第一、二、三作者，干预实施为第一、四、五、六、七作者，结果评估为第一、二作者，均经过系统培训，未使用盲法评估。
方法：
细胞培养[9]：新生鼠在无菌条件下取脑，分离出基底前脑，置于1.25g/L胰蛋白酶中消化，37 ℃ 30 min，加10%小牛血清终止消化，用细嘴吸管反复吹打制成细胞悬液，离心；加入D-Hank’s液5 mL，吹打、离心，反复3遍。用含B27的DMEM/F12培养基6 mL稀释，然后经200目不锈钢筛网过滤使细胞充分分离，成单细胞悬液。取少量悬液样本，加等量4g/L锥虫蓝吹打3次使其均匀混合，用血球计数板在倒置显微镜下计数活细胞，再将单细胞悬液接种于盛有10 mL含B27的DMEM/F12培养基的50 mL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。培养瓶放入37 ℃、体积分数为0.05的CO2饱和湿度培养箱中培养。每天镜下观察，待原代克隆形成后机械分离成单细胞悬液，按上述条件继续培养。以后每5~7 d分离克隆传代1次，方法同前。
巢蛋白抗原的检测：取部分神经球接种到预先置有涂布有0.5 g/L多聚赖氨酸盖玻片的24孔培养板中，按上述条件继续培养3 h后，40 g/L多聚甲醛固定，进行巢蛋白免疫荧光染色。
BrdU标记检测：将BrdU加入传代培养的细胞培养基中，终浓度为6 mg/L，再加入终浓度均为10 μg/L的表皮生长因子和碱性成纤维生长因子按上述条件继续培养2 d。新克隆形成后，将神经球均匀地接种到预先置有涂有 0.5 g/L多聚赖氨酸盖玻片的24孔培养板中，待

神经球贴壁3 h后行免疫荧光染色。
诱导分化及免疫荧光染色：取部分BrdU标记的神经球，用DMEM/F12洗3遍，机械分离成单细胞悬液。取单细胞悬液均匀地接种到预先置有涂布有0.5 g/L多聚赖氨酸盖玻片的24孔培养板中，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10 μg/L的表皮生长因子、碱性成纤维生长因子，按上述条件继续培养，7 d后行免疫荧光染色。

2 结果

2.1 细胞形态观察 原代培养的基底前脑细胞呈透亮的圆球形，悬浮生长。二三天后细胞数目明显减少，视野中可见大量的细胞碎片，部分细胞开始分裂，形成数个细胞的团块，5 d后细胞明显增加，细胞团体积不断增大，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，见图1。球中的细胞形态规则，边界清楚，体积较大，折光性较强，胞浆颜色较深，核/浆比较大。传代的神经干细胞经培养出现与原代培养相同的大量克隆细胞球，悬浮生长，折光性强，细胞形态不变。

2.2 巢蛋白抗原的表达 贴附于盖玻片3 h后，神经球有少许突起从细胞团边缘长出。免疫荧光染色显示，传代神经球中的细胞均呈巢蛋白抗原阳性，见图2，表明培养得到的神经球中的细胞具有胚胎源性。

2.3 BrdU标记检测 加入BrdU标记4 d后可见新的克隆球已经形成，将神经球均匀地接种到预先置有多聚赖氨酸盖玻片的24孔培养板中。待克隆球贴壁后免疫荧光染色显示，新形成的细胞克隆球中的细胞均为BrdU阳性，见图3，表明克隆球是由不断分裂增殖的细胞组成的。

2.4 诱导分化结果 贴壁2 d后可见神经干细胞形态变为不规则，部分长出较短小的突起；1周后突起明显增长，数目增多。神经干细胞免疫荧光染色结果显示，在体积分数为0.1小牛血清诱导下，20%分化成神经元；在终浓度为10μg/L的表皮生长因子诱导下，22％分化为神经元；在终浓度为10μg/L的碱性成纤维生长因子诱导下，40%分化为神经元，见图4。

3 讨论

神经干细胞是指具有自我更新能力，并可增殖分化为神经元、星形胶质细胞和少突胶质细胞的一类细 胞[10-11]。神经干细胞的存在并分离培养成功，为神经系统的发育和中枢神经功能的重建提供了一条新的思路。从神经管的出现、神经组织的分化到脑泡的形成与分化都经历了神经干细胞到成熟神经细胞的过程，研究神经干细胞的增殖、迁移和分化为揭示神经系统的发生、发育过程提供了可靠的依据[12-13]。神经干细胞移植对于神经系统退变性疾病如老年性痴呆、帕金森病等是一种有效的治疗途径，同时对于脑损伤的修复疗效也较好，神经干细胞的发现解决了脑组织供体的不足以及移植细胞与宿主脑组织难以整合的难题[14-15]。近几年来，神经干细胞也是基因治疗神经系统疾病的理想载体细胞[16]。
由神经干细胞增殖而成的细胞球表达神经干细胞特征性的巢蛋白蛋白。巢蛋白蛋白随着神经细胞的迁移和分化开始逐渐消失，在神经分化结束时停止表达，作为神经干细胞的特有标志物[17]。Brdu是一种胸腺嘧啶类似物，在DNA合成时可被细胞摄取利用而参与DNA的组成[18]，因此Brdu是增殖细胞的一种标记物。
许多学者都培养到了神经干细胞，并且研究不同刺激因子对神经干细胞的影响。表皮生长因子和碱性成纤维生长因子对于成年大鼠不同脑区神经干细胞有不同的作用，表皮生长因子对广泛脑区的神经前体细胞都有作用；表皮生长因子和碱性成纤维生长因子分别通过自身受体EGFR和FGFR1起作用，Gritti等已成功地克隆出这两种受体[19]。在表皮生长因子和碱性成纤维生长因子存在的条件下，神经干细胞克隆生长形成神经球，将神经黏附到一定的基质上，能够分化成神经元和神经胶质细胞。然而，表皮生长因子和碱性成纤维生长因子除了作为有丝分裂原在体外促进神经干细胞分裂增殖外，它对神经干细胞的分化有不同的影响[20]。以往观点认为表皮生长因子诱导增殖的神经干细胞大部分向胶质细胞分化，而碱性成纤维生长因子诱导增殖的神经干细胞多数向神经元分化，并且碱性成纤维生长因子对神经干细胞分化的影响则呈浓度依赖性。将外源性表皮生长因子和碱性成纤维生长因子注入大鼠脑内，发现碱性成纤维生长因子促进新生神经元的增加，表皮生长因子则主要促进神经胶质细胞的再生。本实验着重观察表皮生长因子和碱性成纤维生长因子对培养的神经干细胞分化为神经元的影响，用血清作为对照。结果显示在表皮生长因子的诱导下，22%分化为神经元；碱性成纤维生长因子能够使40%神经干细胞分化为神经元。
很多神经营养因子和细胞因子能够促使神经干细胞向神经元分化，如神经生长因子、脑源性神经营养因子、血小板源性生长因子、胰岛素样生长因子等则可以诱导神经干细胞向神经元方向分化，促进神经元树突的生长及神经营养作用。联合不同的因子甚至能够使神经干细胞定向分化，碱性成纤维生长因子、肝素和层粘连蛋白联合应用能够诱导神经干细胞分化为多巴胺能神经元[21]；脑内注射丝裂原、视黄酸和神经生长因子能够原位诱导神经干细胞分化为胆碱能神经元[22-23]。基底前脑神经干细胞的定向分化能力如何？进一步的研究正在进行中。

4 参考文献

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