摘要
目的 :观察大鼠神经干细胞的体外生长特性。方法 :利用无血清培养基悬浮培养 ,观察神经干细胞的体外生长过程。并比较不同接种密度 ,不同传代时间对神经干细胞生长的影响。用免疫细胞化学技术检测神经干细胞巢蛋白 (nestin)的表达 ;用吖啶橙 (AO) /溴化乙啶 (EB)检测细胞活性 ;用DAPI荧光染料标记细胞。结果 :神经干细胞聚合成细胞团生长 ,细胞团成分复杂。细胞接种密度以 5× 1 0 5个细胞 /ml组生长较好 ;原代细胞生长 3～ 4d后传代为宜。传代后 4d时可获得大量nestin阳性细胞。DAPI标记率为 1 0 0 %。结论 :细胞团成分复杂。神经干细胞要高密度接种 ,及时传代。
Objective: To observe the growth characteristics of in vitro cultured neural stem cells isolated from new-born rats. Methods: Serum-free suspension culture was used to observe the growth of neural stem cell. The effects of feed density and passage time on the cell growth were investigated. Immunocytochemistry staining was preformed to detect nestin antigen of the cells. Acridine orange (AO) / ethidium bromide (EB) staining was applied to detect the cell vitality, and DAPI fluorescence was used to mark the cells. Results: Rat neural stem cells grew into cell clusters. There were different kinds of cells in the cluster. The cell growth state of feeding with 5×10 5/ml was better than that of 1×10 5 cells/ml. Subculture of the cells at 3-4 d after primary culture was found to grow well. It was revealed that more nestin-positive cells could be obtained on the 4th day after subculture. The DAPI could mark the cells nearly 100%, which still could be observed after continuing culture for one month. Conclusion: There are different kinds of cells in the cluster. It is recommend that rat neural stem cells should be fed in high density and subcultured them in time. DAPI can be used as an effective marker in vitro.
出处
《解剖学杂志》
CAS
CSCD
北大核心
2004年第6期634-638,F002,共6页
Chinese Journal of Anatomy
