新生大鼠全大脑组织分离诱导分化神经干细胞的体外实验

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1~3 d 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。

Isolation and differentiation of neural stem cells from neonatal rats in vitro

BACKGROUND: Neural stem cells are always derived from fetal rats and adult rats, and it is complex to isolate the cells by cell culture. OBJECTIVE: To study a convenient and effective method for the isolation and the culture of neural stem cells in neonatal rats. DESIGN, TIME AND SETTING: An observation study based on cells was carried out in the Chongqing Medical University (Chongqing, China) from October 2006 to March 2007. MATERIALS: Wistar neonatal rats of 1-3 days old. METHODS: Subsequent to trypsin digestion, primary culture of the cells was performed in serum-free suspension culture medium. Then the cells were induced to incubate in DMEM/F12 containing 0.10 volume fraction of fetal bovine serum. MAIN OUTCOME MEASURES: Phase contrast microscopy was employed to observe the growth of neural stem cells and the morphology of the differentiated cells. Neural stem cells and the differentiated neurons were identified using indirect immunofluorescence cytochemistry, as well as expression of gilal fibrillary acidic protein. Moreover, the proliferation of the BrdU-labeled neural stem cells was also investigated. RESULTS: The neural stem cells isolated from neonatal rat brains had the potential of serial passage and proliferation, besides, they express neuroepithelial stem cell protein (nestin) and differentiate into neurons, astrocytes and oligodendrocytes. CONCLUSION: Neural stem cells can be harvested from neonatal rat brains at a large scale, and they maintain their undifferentiated features and have the capacity of self-renewal and pluripotentiality.