两种方法纯化胎鼠脑室管膜下区神经干细胞的结果比较

Comparison of efficacy of two purified methods for neural stem cells from the subependymal zone of embryonic rat brain

目的利用有限稀释法和连续传代法纯化体外培养的神经干细胞(neural stem cells,NSCs),并对2种方法纯化后的细胞纯度及增生能力进行比较。方法机械分离孕龄14.5d的胎鼠脑室管膜下区细胞,利用有限稀释法和连续传代法纯化NSCs,通过免疫细胞化学法鉴定纯度(Nestin阳性率)及分化细胞的类型。将2种方法纯化后的第3代NSCs以相同的密度接种后连续培养6周,每周进行细胞计数,绘制生长曲线。结果有限稀释法和连续传代法纯化NSCs的Nestin阳性率均高于原代NSCs(31.00%±0.02%),有限稀释法的Nestin阳性率(91.00%±0.03%)高于连续传代法(58.80%±0.02%,P<0.05)。有限稀释法纯化NSCs的生长曲线一直处于增生趋势,而连续传代法的细胞前3周是上升趋势,以后趋于平缓(P<0.05)。结论有限稀释法能有效纯化体外培养的NSCs,并保持细胞良好增生能力及多向分化潜能,为基因治疗视神经损伤疾病提供大量种子细胞。

Objective To compare the cell purity and productive activity after purifying neural stem cells （NSCs） by limiting dilution assay and consecutive passage assay. Methods NSCs were separated mechanically from the subependymal zone of 14.5 days embryonic rat brain, and purified by limiting dilution assay and consecutive passage assay separately. Immunocytochemical techniques were carried out to identify the purity （ Nestin positive rate） and types of differentiated cells. The third passage cells purified with 2 methods were seeded in same density and then cultured for 6 weeks. Cells were counted every week, and growth curves were drawn. Results The Nestin positive rate of NSCs in both limiting dilution assay and consecutive passage assay groups were higher than that of primary NSCs （ 31.00% ±0. 02% ）, and the former （91.00% ±0. 03% ）was higher than the latter （58.80% ± 0.02% ,P 〈 0.05 ）. The cell growth curves of the purified NSCs with limiting dilution assay displayed a constant multiplication trend, and the other group showed a rising tendency in former 3 weeks and then grew slowly（P 〈 0.05 ）. Conclusion Limiting dilution assay can effectively purified NSCs in vitro, and maintains them with favourable reproductive activity and multi-directional differentiated capability, which provides a large number of seed cells for treating optic nerve injury with gene therapy.