Culture and biological characteristics of neural stem cells from caveolin-1 knockout embryonic mice

doi:10.3969/j.issn.2095-4344.2014.23.022

Abstract

Abstract:

BACKGROUND: Caveolin-1 is expressed in mammalian brain and involved in the normal development of the brain, which can affect the proliferation of neural stem cells in the brain.

OBJECTIVE: To acquire neural stem cells from caveolin-1 knockout embryonic mice in vitro and study their biological characteristics.

METHODS: The whole brain was separated from C57BL/6 mice and caveolin-1 knockout C57BL/6 mice respectively at encyesis 14-16 days. Single cell suspension was obtained by enzyme digestion, and cultured in the conditioned medium of neural stem cells. Following 7 days of primary culture, the cells were induced in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10% fetal bovine serum for 7 days.

RESULTS AND CONCLUSION: The major cells of the cell suspensions from the fetal mouse brain were dead at 1 day after culture, and some single cells floated in the medium and their transmittance were better, and then they gradually formed multicellular balls after 3 days. A small amount of cells were adhered at the bottom of culture plate after passage, and a great amount of cell balls appeared after 7 days. The proliferation rate of neural stem cells from caveolin-1 knockout mice was higher than that from normal mice. The cell balls were nestin-positive and their differentiated cells was positive for neurofilament 200, glial fibrillary acidic protein or O4, respectively. All of the cells from normal mouse brain were positive for caveolin-1, but the cells from caveolin-1 knockout mice were negative for caveolin-1 by immunocytochemistry. Moreover, the speed of cell ball formation and the number of cell balls in neural stem cells from caveolin-1 knockout mice were better than those from normal mice. Caveolin-1 negative neural stem cells were cultured successfully from caveolin-1 knockout mouse brain, and the results show that caveolin-1 can promote the proliferation of neural stem cells and inhibit their differentiation in vitro.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, neural stem cells, caveolins

CLC Number:

R394.2

Liu Bai-yan, Yu Yue, Yi Jian, Chen Xue-mei, Cai Guang-xian. Culture and biological characteristics of neural stem cells from caveolin-1 knockout embryonic mice[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(23): 3739-3744.

Figures/Tables 2

2.1 神经干细胞生长情况观察结果两种来源细胞悬液接种1 d后，镜下可见较多细胞发生死亡，表现为透光度较差的小点，其间可见单个细胞漂浮于培基中，部分为数个细胞的聚集，和周围悬浮细胞碎片相比，呈透光度较好的亮点。原代培养两三天后，悬浮细胞碎片间可见一定数量的细胞聚集，形态较为规则，多为类圆形或近圆形，胞体通透，呈悬浮生长，第1次换液后，悬浮细胞碎片较之前明显减少，神经干细胞生长迅速，可见有较多细胞团聚集，大小不一，形态各异，呈球型、长椭圆型或桑葚型悬浮生长。采用accutase消化加机械吹打的方式传代后，可见呈悬浮生长的单细胞逐渐形成球形克隆，成球速度快于原代培养，同时可见培养板底部细胞部分发生分化(图1)。 2.2 神经干细胞的鉴定结果免疫细胞化学检测显示，附着于多聚赖氨酸包埋盖玻片上的细胞球均呈巢蛋白抗原强阳性(图2)。 2.3 诱导分化细胞鉴定结果加入血清诱导2 d后，两组可见细胞球贴壁，细胞团周围长出长短不一的突起，7 d中小细胞球基本铺展开，部分细胞移行至一定距离，细胞呈胞体形态不一带突起的细胞。免疫细胞化学检测显示，两组细胞可分别呈神经丝蛋白200、胶质纤维酸性蛋白和O4抗原阳性(图2)。 2.4 细胞细胞质膜微囊蛋白1表达鉴定来源于正常胎鼠的神经干细胞和分化细胞均呈细胞质膜微囊蛋白1阳性表达，而来源于细胞质膜微囊蛋白1敲除胎鼠的神经干细胞和分化细胞细胞质膜微囊蛋白1表达均呈阴性(图3)。 2.5 细胞质膜微囊蛋白1对神经干细胞增殖的影响采用克隆球计数的方法对两种来源胚胎神经干细胞进行计数发现，细胞质膜微囊蛋白1敲除胎鼠神经干细胞成球速度和成球数量优于正常胎鼠神经干细胞，两组比较差异有显著性意义(P < 0.05，图3，表1)。"

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