摘要
背景:随着神经干细胞培养技术条件的成熟,为颅脑损伤后神经系统功能重建、神经再生和神经系统疾病治疗提供了新的思路和途径。但移植所用的细胞来源及移植后的神经干细胞能否分化为神经细胞是急需解决的重要问题。目的:拟将体外分离培养的鼠胚胎神经干细胞稳定增殖传代,并诱导分化出神经系统的3种基本细胞。设计、时间及地点:细胞学体外观察,于2007-06/2008-06在新疆维吾尔自治区人民医院完成。材料:14～16d孕龄Wistar大鼠胚胎,由新疆自治区实验动物研究所和新疆医科大学实验动物中心提供。方法:取胎鼠额叶大脑皮质,组织块消化法体外分离培养神经干细胞,在表皮生长因子、碱性成纤维生长因子和B27联合作用下使其稳定增殖。原代培养7d后,加入含体积分数为10%胎牛血清的DMEM/F12培养基诱导14d。主要观察指标:倒置显微镜下观察神经干细胞的生长形态,并行巢蛋白免疫荧光染色鉴定。应用免疫组化染色检测诱导后神经干细胞的分化情况。结果:原代培养一两天细胞散在分布,胞体较小,呈圆形,折光性较好,3d后逐渐形成由数个细胞组成的细胞球;传代后贴壁细胞多数分化为长梭形或扁平形的胶质细胞,10d时可见大量由数十至数百个细胞组成的较大细胞球,其生长速度基本与原代培养相同,但成球速度明显加快;免疫荧光染色呈巢蛋白阳性反应。以含体积分数为10%胎牛血清的培养液诱导后,免疫化学染色示神经干细胞球呈神经元特异性烯醇化酶、胶质纤维酸性蛋白、髓鞘碱性蛋白阳性表达。结论:实验成功从鼠胚胎脑皮质分离培养出神经干细胞,并诱导分化为神经元细胞、星形胶质细胞和少突胶质细胞。
BACKGROUND： The maturity of culture technique of neural stem cells （NSCs） provides a new ideal and pathway of reconstruction of the nervous system, neural regeneration and nervous system diseases following craniocerebral injury. However cell source, whether NSCs can differentiate into neural cells are urgent problems. OBJECTIVE： To induce the stable proliferation and passage of in vitro-separated cultured mouse embryonic NSCs, and to differentiate into more than 3 forms of cells in the nervous system. DESIGN, TIME AND SETTING： The cytology in vitro study was performed at the People＇s Hospital of Xinjiang Uygur Autonomous Region from June 2007 to June 2008. MATERIALS： Embryoes of Wistar rat of gestation for 14-16 days were supplied by the Animal Experimental Institute of Xinjiang Uygur Autonomous Region and Animal Experimental Center of Xinjiang Medical University. METHODS： The neural stem cells were derived from the cortex tissue of fetal rats by the digestion method to keep the stable proliferation of NSCs under the combination of epidermat growth factor, basic fibroblast growth factor and B27. Following 4 days primary culture, NSCs were incubated in DMEM/F12 containing fetal bovine serum of 10% volume fraction for 14 days. MAIN OUTCOME MEASURES： NSC morphology was observed with an inverted microscope. Nestin staining was performed. Differentiation of NSCs was detected following induction using the immunohistochemistry. RESULTS： Following 1 or 2 days primary culture, NSCs were round, scattered, with small bodies and good refraction. Following 3 days, several NSCs formed cell spheres. After passage, adherent cells mostly were spindle or flat glial cells. At day 10, abundant cells formed large cell spheres, and its growth speed was identical to primary cultured cells. Immunofluorescence staining showed NSCs were positive Nestin. Following induction of fetal bovine serum of 10% volume fraction. Immunochemistry staining demonstrated that NSC spheres exhibited a positive reaction for neuron specific enolase, glial fibrillary acidic protein and myelin basic protein. CONCLUSION： The neural stem cells are derived from the cortex tissue of rat embryo and differentiate into neurons, astrocytes and oligodendrocytes.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第19期3651-3655,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
新疆维吾尔自治区"十一
五"科技攻关和重点科技项目(200633128(2))
新疆维吾尔自治区卫生厅青年科技人才专项科研基金(2007Y26)~~
