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背景:神经干细胞的供体一般以胎鼠和成年鼠为主,利用细胞培养技术分离步骤较繁琐。目的:以新生大鼠为神经干细胞供体,拟建立一种较为简便、细胞获得率较高的分离培养方法。设计、时间及地点:以细胞为对象观察性实验于2006-10/2007-03在重庆医科大学完成。材料:新生1～3d的Wistar大鼠全大脑。方法:以胰蛋白酶消化、无血清、悬浮培养原代细胞,并加含体积分数为0.10胎牛血清的DMEM/F12培养液诱导其分化。主要观察指标:应用相差显微镜观察神经干细胞的生长特点及分化后的细胞形态学变化。应用间接免疫细胞化学染色法鉴定神经干细胞及其分化后神经元和胶质细胞标志蛋白的表达。以BrdU标记神经干细胞,观察其增殖情况。结果:新生大鼠脑组织分离的细胞具有连续传代和增殖的能力,能稳定表达神经干细胞特异性巢蛋白。诱导分化后的细胞能表达神经元细胞、星形胶质细胞、少突胶质细胞的特异性蛋白。结论:从新生大鼠脑组织分离培养出的神经干细胞获得率高,保持了干细胞的未分化属性,具有自我更新和多项分化潜能。
Background: Donors of neural stem cells are mainly fetus and adult mice, and the steps of cell culture are more complicated. OBJECTIVE: To establish neonatal rat as a neural stem cell donor, and to establish a simple and convenient method for isolation and culture with high cell yield. DESIGN, TIME AND SETTING: The observation experiment with cells was performed at Chongqing Medical University from October 2006 to March 2007. MATERIALS: Wistar rats whole brain of newborn 1 ~ 3d. Methods: Primary cells were cultured with trypsin digestion, serum-free and suspension culture, and cultured in DMEM / F12 medium containing 0.10% fetal bovine serum. MAIN OUTCOME MEASURES: The growth characteristics of neural stem cells and morphological changes after differentiation were observed by phase contrast microscopy. Indirect immunocytochemical staining was used to identify neural stem cells and their differentiated neuronal and glial cell marker proteins. BrdU labeled neural stem cells, observe the proliferation. Results: The cells isolated from brain tissue of neonatal rats had the ability of continuous passage and proliferation, and could stably express neural stem cell-specific nestin. The differentiated cells can express specific proteins of neurons, astrocytes and oligodendrocytes. CONCLUSION: Neural stem cells isolated and cultured from neonatal rat brain have high yield, maintain the undifferentiated nature of stem cells, and have the potential of self-renewal and differentiation.