新生大鼠神经干细胞的分离、培养和鉴定

Isolation,Cultivation and Identification of Neonatal Rat Neural Stem Cells

目的获得新生大鼠神经干细胞,为深入研究其增殖分化奠定基础。方法从新生24小时内大鼠端脑及中脑部位分离NSC,采用体外无血清悬浮培养,以获得能在体外长期生存的NSC;通过单细胞克隆实验检测其自我更新能力,免疫细胞化学检测NSC标志物nestin表达及分化为神经元和胶质细胞的能力;用细胞计数法研究其生长特点并绘制生长曲线。结果获得的NSC能在体外长期生存,具有很强的增殖能力、自我更新能力,能分化为神经元和胶质细胞,具有多向分化能力,表达nestin,体外传代至10代细胞数量扩增了40倍。结论成功地从新生大鼠端脑和中脑中分离得到了能在体外长期培养的NSC。

Objective To obtain neonatal rat neural stem cells （NSCs） for further study on its proliferation and differentiation. Methods To isolate the primary NSCs from endbrain and midbrain of neonatal rats （ in 24h after birth） , the cells are suspended and cultured in serum free medium （containing bFGF and EGF） for long-term survival in vitro. Technique of single-cell clone culture was used to confirm the character of self-renewing. Ability of multi-directional differentiation and specific antigen, nestin, were detected by immunocytochemistry. Cytometry was used to evaluate the proliferation capability of NSCs and the growth curve is plotted. Results NSCs can survive in vitro for long time. They show muhi-potential of differentiate into neurons and glial cells and the ability of self-renewing and proliferating. The NSCs are positive for nestin expression. The total number of the cell expands 40 folds after 10 times passage. Conclusion The NSCs are isolated from endbrain and midbrain of neonatal rats. These cells can be cultured for long time in vitro.