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背景:神经干细胞作为修复损伤神经组织的细胞源已引起学者的关注,如何有效的获取更多的神经干细胞逐渐成为重点解决的课题之一。目的:观察大鼠室管膜下区和海马回神经干细胞体外培养、传代和分化情况。设计:单一样本实验。单位:中山大学附属第三医院神经外科。材料:取出生后3dSD大鼠10只,雌雄不拘,由中山大学实验动物中心提供。巢蛋白抗体(兔抗大鼠)、神经微丝蛋白(NF-200)抗体(兔抗大鼠)、胶质纤维酸性蛋白抗体(小鼠抗大鼠)均购自Sigma公司。方法:实验于2006-09/12在中山大学基础医学院组织胚胎学实验室完成。实验过程中对动物的处置符合动物伦理学要求。从新生大鼠海马、室管膜下区分离神经干细胞,采用DMEM/F12无血清悬浮的条件培养,同时在培养基中加入碱性成纤维细胞生长因子和表皮生长因子代替血清中含有的分裂原,进行体外扩增培养、传代观察。主要观察指标:采用免疫荧光检测技术,通过检测神经干细胞表达的nestin、神经元细胞表达的神经微丝蛋白和星形胶质细胞表达的胶质纤维酸性蛋白,鉴定神经干细胞及其分化结果。结果:分离获取的细胞具有自我更新和增殖能力,原代及传代培养20代后,在倒置显微镜下仍保留典型的“细胞克隆球”特征,神经克隆球通过nestin免疫荧光染色,呈阳性反应。经诱导后,细胞克隆球细胞外迁贴壁生长,可分化为神经微丝蛋白阳性的神经元细胞和胶质纤维酸性蛋白阳性的星形胶质细胞。结论:实验分离培养出具有自我更新和增殖能力的神经干细胞,其可诱导分化为神经元样细胞和星形胶质细胞。
BACKGROUND: Neural stem cells, as the cell source for repair of damaged nerve tissue, have drawn the attention of scholars. How to effectively obtain more neural stem cells has gradually become one of the key problems to be solved. OBJECTIVE: To observe the in vitro culture, passage and differentiation of rat NSCs and hippocampal neural stem cells. Design: Single sample experiment. Unit: Department of Neurosurgery, Third Affiliated Hospital of Sun Yat-sen University. MATERIALS: Ten SD rats were randomly divided into male and female rats, which were provided by Experimental Animal Center of Sun Yat-sen University. Nestin antibodies (rabbit anti-rat), neurofilament protein (rabbit anti-rat), glial fibrillary acidic protein antibody (mouse anti-rat) were purchased from Sigma. Methods: The experiment was performed at the Embryology Laboratory of School of Basic Medicine, Sun Yat-sen University from September to December 2006. The handling of animals during the experiment conformed to the ethical requirements of animals. Neural stem cells were isolated from the hippocampus of neonatal rats and subependymally and cultured in DMEM / F12 serum-free medium. At the same time, basic fibroblast growth factor and epidermal growth factor were added into the medium instead of the mitogen , In vitro expansion and culture, passage observation. MAIN OUTCOME MEASURES: Neural stem cells and their differentiating results were identified by immunofluorescence assay, detection of nestin expressed by neural stem cells, neurofilament expressed by neurons and glial fibrillary acidic protein expressed by astrocytes. Results: The isolated cells had self-renewal and proliferative ability. After 20 generations of primary culture and subculture, the typical “cell clone spheres” were retained under an inverted microscope. The neuronal clone spheres were positive by nestin immunofluorescence staining reaction. After induction, the cell colonies migrate adherently adherently and grow into neurons that differentiate into neurofilament-positive neurons and glial fibrillary acidic protein-positive astrocytes. CONCLUSION: Neural stem cells with self-renewal and proliferation ability were isolated and cultured in vitro, which can be induced to differentiate into neuron-like cells and astrocytes.