生理学报Acta Physiologica Sinica, June 25 2003 55(3)
研究论文
胚胎大鼠脑和脊髓神经干细胞的分离和培养
富赛里1, 马政文1, 尹岚1, 陆佩华1，, 徐晓明1,2
1上海第二医科大学神经生物学实验室, 上海 200025;
2美国肯塔基脊髓损伤研究中心, 美国路易斯威尔大学神经解剖实验室, 肯塔基州 40292, 美国
摘要: 本研究采用显微解剖、 无血清细胞培养和免疫荧光细胞化学染色等实验技术, 成功地建立了胚胎大鼠脑和脊髓神经干细胞(NSCs)的分离和培养方法。结果显示, (1)在含成纤维细胞生长因子-2 (FGF-2)和表皮生长因子(EGF)的无血清培养液中, 两种来源的NSCs经体外培养8－10代后, 其细胞数呈指数级增加, 其中脑来源的NSCs数由原代培养时的1×106增加至1×1012, 脊髓来源的NSCs数从1×106增加至1×1011。增殖的细胞表达神经上皮干细胞蛋白(nestin); (2)在含1%胎牛血清(FBS)的培养条件下, 它们都能被诱导分化为神经元、 少突胶质细胞和星型胶质细胞。但其分化比例可随细胞传代次数的增加而改变, 其中, 大脑来源的NSCs分化为神经元的比例从第二代(P2)的11.95±2.5%下降至第五代(P5)的1.97±1.16% (P<0.01), 而少突胶质细胞的分化比例则基本保持不变, 这一分化格局同样可在脊髓来源的NSCs中发现。结果表明, 我们所分离和培养的细胞在体外经多次传代后仍具有很强的增殖能力和多向分化潜能, 它们都表达nestin, 属于中枢神经系统的干细胞。
关键词: 中枢神经系统; 神经干细胞; 胚胎; 成纤维细胞生长因子－2; 表皮细胞生长因子
中图分类号: Q78
Isolation and
cultivation of neural stem cells from the embryonic rat brain and spinal cord
FU Sai-Li1, MA Zheng-Wen1, YIN Lan1, LU Pei-Hua1, XU Xiao-Ming1,2
1Department of Neurobiology, Shanghai Second Medical University, Shanghai 200025;
2Kentucky Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville, 511S. Floyd Street MDR616, Louisville, KY40292, USA
Abstract: The aim of this study was to establish the culture system of isolation and cultivation of the neural stem cells (NSCs) from the embryonic rat brain and spinal cord. The methods of microscopic dissection, cell culture and immunofluorescence cytochemistry were used. The results are as follows. (1) In the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF), both brain- and spinal cord-derived stem cells proliferated and expanded in vitro for 8－10 passages (over 60 d). The period of expansion resulted in a 106- fold increase in brain-derived NSCs and 105- fold increase in spinal cord-derived NSCs. These proliferating cells expressed nestin. (2) In the medium containing 1% FBS, the two NSCs populations could be induced to differentiate into neurons, astrocytes and oligodentrocytes. The percentage of neurons (β-tubulinⅢ-ir) differentiated from brain-derived NSCs decreased rapidly from 11.95±2.5% at passage 2 (P2) to 1.97±1.16% at passage 5 (P5). Significant difference was shown between P2 and P5 (P<0.01). The percentage of oligodentrocytes (Rip-ir) differentiated from brain-derived NSCs remained mostly unchanged from 8.66±2.93% at P2 to 9.12±1.13% at P5. The same differentiation patterns were found in spinal cord-derived NSCs. All these results indicate that both embryonic rat brain and spinal cord-derived NSCs can expand in vitro through multiple passages, and retain the capacity to differentiate into all three major l types of cells in the central nervous system.
Key words: central neural system; neural stem cell; embryo; fibroblast growth factor-2; epidermal growth factor