人脑神经干细胞对不同储存方式及分离培养和诱导分化条件的要求(英文)

Requirements of human brain-derived neural stem cells for different storage,isolation and cultivation

背景:近年来发现神经干细胞可于体外增殖并分化为神经细胞,是用来修复替代损伤神经组织的较为理想的来源,但是神经干细胞的培养中如何提高它的增殖能力,诱导分化成需要的表现型,尚处于研究探讨阶段。目的:探讨细胞冻存和非冻存方法分离培养人脑神经干细胞及诱导其向成熟神经细胞分化的培养条件。设计:以细胞为观察对象,单一样本研究。单位:江西医学院泌尿外科研究所,江西医学院第二附属医院神经外科。材料:实验于2003-12/2004-06在江西医学院泌尿外科研究所完成。取16周龄新鲜人胚脑组织。方法:用胰蛋白酶消化法从人胚脑中分离单个细胞,细胞冻存1个月后复苏或/和新鲜细胞用无血清培养基进行体外培养,碱性成纤维细胞生长因子和表皮生长因子刺激细胞生长,用血清诱导其分化;采用免疫荧光细胞化学染色方法检测培养的细胞神经巢蛋白抗原和分化后成熟神经细胞抗原神经元特异性烯醇化酶的表达。观察碱性成纤维细胞生长因子和表皮生长因子及血清对分离培养的人脑神经干细胞增殖与分化的影响。主要观察指标:①冻存与新鲜细胞后续培养的生长状况。②细胞形态变化。③神经干细胞标志物神经巢蛋白及成熟神经细胞标志神经元特异性烯醇化酶的鉴定。结果:①从胚龄16周的新鲜人胚脑中成功分离出神经干细胞。人胚脑原代细胞为圆形亮球状,培养3d后可见细胞开始聚集成神经球,部分呈悬浮生长,2周后神经球增大,部分细胞增大数倍,具有极强折光性,可见分裂增殖。经传代后细胞仍保持原有特征,细胞形态不变。克隆细胞在有血清培养基中培养,24h后可见大部分细胞贴壁生长,细胞从神经球游出分散,形态不规则;48h后多数细胞分化为大量形态不一的、分散成片的多突起星状细胞和神经元样细胞,突起相互交织成网。②分离出的神经干细胞在体外培养条件下传代培养,并表达神经干细胞标志巢蛋白;含血清培养基培养48h分化后的细胞主要表达成熟神经细胞标志神经元特异性烯醇化酶。③冻存复苏的细胞95%以上为活细胞,与新鲜细胞比较,细胞生长状况无明显差别。结论:用无血清培养条件下结合碱性成纤维细胞生长因子和表皮生长因子的作用,使干细胞体外得到了分离培养、增殖和纯化,证实其方法可行,同时冻存和新鲜细胞的比较,说明可用冻存方法保存人胚脑细胞,复苏后使用。

BACKGROUND:Due to a new finding in the in vitro proliferation and differentiation of neural stem cells,neural stem cells are regarded as optimal materials for repairing and replacing the injured neural tissues.But how to enhance the proliferation of neural stem cells and how to induce neural stem cells to differentiate into certain phenotypes remain objects for discussing. OBJECTIVE:To investigate the frozen/nonfrozen storage techniques in isolation and cultivation of neural stem cells isolated from human brain and to discuss the conditions suitable for their differentiation. DESIGN:A single sample based on cells. SETTING:Urinology Institute of Jiangxi Medical University and Neurosurgery Department of Second Hospital Affiliated to Jiangxi Medical College. MATERIALS:From December 2003 to June 2004,this study was conducted at the Urinology Institute of Jiangxi Medical College.Brain tissues removed from sixteen-week-old embryos were studied. METHODS:After being digested with trypsin,cells were separated from the brains of embryos.They were cultured in the serum-free media,stimulated by basic fibroblast growth factor and epidermal growth factor and were induced to differentiate using serum.Immunofluorescent cytochemical staining technique was adopted to detect the expressions of the Nestin and the neuron-specific enolase(the marker for mature neural cells) in these cells.The influence of basic fibroblast growth factor,epidermal growth factor and serum on the proliferation and differentiation of the neural stem cells were investigated. MAIN OUTCOME MEASURES:The main outcome measurements in this study were:①living status of the freeze-thaw neural stem cells and the fresh isolated neural stem cells,②morphological changes in these cells,③identification of the Nestin and the neuron-specific enolase in these cells. RESULTS:①Neural stem cells were isolated from fresh 16-week-old embryo brain successfully.The embryo brain cells in primary culture were clear and round.After 3 days some cells aggregated to form neurospheres and some others suspended.Two weeks later,the neurospheres enlarged and a part of cells in it expanded into folds with strong reflection and proliferation.The continuous cultured cells inherited these characteristics and kept the morphological properties.The clones were cultured in the medium with serum.Twenty-four hours later,most of the cells were attached to the bottom of the plates.Cells were dissociated from the neurospheres and became irregular.Forty-eight hours later,most of the cells differentiated into many scattered patches of astrocytes with various shapes and neurites and many neuron-like cells.The neurites formed an interconnected network.②The isolated neural stem cells were continuously cultured in vitro and they expressed Nestin,the marker of neural stem cells.After being cultured in medium with serum for 48 hours,the differentiated cells mainly expressed neuron-specific enolase,the marker of mature neural cells.③95%of the freeze-thaw cells were alive.Their living status had no significant difference compared to that of the fresh isolated cells. CONCLUSION:Serum-free culture medium,together with basic fibroblast growth factor and epidermal growth factor,enables the in vitro culture,proliferation and purification of the neural stem cells.The feasibility of the method is confirmed.Meanwhile,the comparison between the cultures of freeze-thaw cells and fresh isolated cells indicated that frozen storage can be used as a way to preserve the human embryo brain cells,which will be available for study after thaw.