胎鼠纹状体神经干细胞分离培养及鉴定

Isolation,culture and identification of neural stem cells from fetal rat striatum

背景:从胎鼠神经系统的多个部位如大脑半球、海马、皮质、脑室区等可分离出神经干细胞。目的:探讨胚胎大鼠大脑纹状体神经干细胞的取材、分离、培养和鉴定方法,观察神经干细胞增殖、传代和分化的规律。设计、时间及地点:观察性实验,于2008-03/08在烟台毓璜顶医院中心实验室完成。材料:选用普通级健康成年Wistar大鼠12只,按雌雄1∶1比例于每晚6时合笼,次晨检查出阴道栓子(阴道内皮结晶)为妊娠0d。方法:在无菌条件下取孕13d胚胎大鼠脑纹状体组织,用无血清培养技术在体外进行神经干细胞的培养、扩增和传代。分别用抗巢蛋白抗体、抗微管相关蛋白2抗体和抗胶质原纤维酸性蛋白抗体对培养的细胞进行干细胞特性和多分化潜能的免疫组织化学鉴定。主要观察指标:①显微镜下观察细胞生长情况。②神经干细胞分化情况及免疫鉴定。结果:①原代细胞种植后24h可见大量分裂期的神经干细胞,呈对称或不对称分裂,细胞核较大,连续观察可见细胞分裂成2个完全独立的神经干细胞;48h后出现许多由4～10个细胞疏松连接的细胞团;神经干细胞进入快速增殖期5d后出现许多大小不等的细胞集落,即神经球,神经球呈规则的圆球状,细胞排列紧密,表面光滑没有突起。②培养4～6h后悬浮的神经干细胞球逐渐贴壁,大部分细胞分化后胞体呈圆形或椭圆形,具有1个或2个长突起,少量细胞具有多个粗长突起。贴壁分化3d左右,具有1个或2个突起的神经元样细胞逐渐减少,具有多个粗长突起的星形胶质细胞样细胞逐渐增多。悬浮的神经干细胞球经巢蛋白染色呈阳性反应,部分细胞呈微管相关蛋白2和胶质原纤维酸性蛋白染色阳性。结论:从胎鼠纹状体中分离培养的神经干细胞具有自我增殖、自我更新能力,并能分化为神经元及神经胶质细胞。

BACKGROUND： Neural stem cells （NSCs） can be isolated from many sites of the nervous system of fetal rats, such as cerebral hemisphere, hippocampus, cortex and ventricle. OBJECTIVE： To investigate the methods of collection, isolation, cultivation and determination of neural stem cells from the striatum of fetal rats, and to observe the proliferation, passage and differentiation of neural stem cells. DESIGN, TIME AND SETTING： This observation experiment was performed at the Center Laboratory of Yantai Yuhuangding Hospital between March and August 2008. MATERIALS： Twelve healthy Wistar rats （male： female at 1：1） mated at 6：00 pm every day, and female rats which were judged pregnant for o day in the following morning when vaginal suppository was used. METHODS： NSCs were sterilely isolated from rat embryonic striatum tissue （E13） and were cultured, amplified and passaged in serum-free medium in vitro. The cells were separately immunostained with anti-Nestin antibody, anti-microtubule-associated protein-2 （MAP-2） antibody and anti-glial fibrillary acidic protein （GFAP） antibody to determine the phenotype of cultured cells, MAIN OUTCOME MEASURES： Growth condition of NSCs was observed under microscope; cell differentiation and identification were studied by immunological methods. RESULTS： In the dividing phase, considerable primary NSCs divided asymmetrically or symmetrically after 24 hours of implantation, with big nuclei. Continuous observation showed that one NSC split into two independent cells. Loosened clonal clusters of cells （4-10 cells/clone） appeared after 48 hours. Colonies, i. e. neurospheres, appeared at various sizes at 5 days following NSCs entered rapid proliferation phase. These floating neurospheres displayed a spherical shape, and the cells arranged closely, with smooth surface, without process. Suspended NSCs presented adherence after 4 6 hours. Most cells became round or oval, and erupted one or two processes and few of them had many thick long processes. Neuronal cells （one or two processes） decreased and astrocytes increased （more than two processes） 3 days later. The neurosphere could express Nestin, generate MAP2- or GFAP-positive neural cells. CONCLUSION： Neural stem cells isolated from fetal rat striatum have the potential of self-proliferation and self-renewal, and can differentiate into neurons and gliocytes.