机械分离与胰酶消化分离对长期培养的神经干细胞神经发生能力的影响

Effect of mechanical and enzymatic dissociation on neurogenesis of long-term expanded neural stem cells

目的探讨神经干细胞（NSCs）培养的分离传代技术及其在长期培养条件下的神经发生能力。方法取孕14d的Wistar胎鼠大脑皮层，消化分离后，以1×10^5／ml浓度进行NSCs原代培养。分别采用机械分离与胰酶消化法进行NSCs传代培养，台盼蓝活细胞计数测定分离后细胞存活率，计算细胞倍增时间和细胞增殖率。取第4、8、12、20、30代机械分离传代培养的NSCs球接种到24孔培养板中的盖玻片上，加入分化培养液，观察长期培养条件下NSCs的神经发生能力的变化。结果与胰酶消化法进行的NSCs球传代相比，机械分离的NSCs球为小细胞球和单细胞传代，传代后细胞存活率高，细胞倍增时间短，增殖能力强（P均〈0．05）。体外长期培养条件下，随着培养时间的延长，NSCs分化为神经元的比例逐渐降低，星形胶质细胞的比例逐渐增高；在4—12代NSCs分化为神经元的能力表现出下降的趋势，但仍较为稳定；12代后，神经元的分化比例迅速下降，星形胶质细胞的比例迅速上升。结论与胰酶消化法相比，逐步减小吸管口径的机械分离传代法，减少了传代对细胞的损伤，保证了大部分细胞间连接的完整性，显著增强了NSCs的增殖能力。体外长期扩增的NSCs，由于微环境和（或）自身基因的调控，随着传代的延续，其神经发生能力逐渐降低，分化为神经元的比例逐渐减少，分化为星形胶质细胞的比例逐渐增加。

Objective To explore the culture method of rat embryonic neural stem cells and the ability of neurogenesis after being cultured long in vitro. Methods Brain tissues were obtained from the forebrain of embryonic 14-day-old Wistar rat embryos. After dissociated by adding 0.25% trypsin and 0.02% ethylene-diamine tetraacetic acid （EDTA） ,the cells were inoculated into preinculated NSCs medium at 1.0 × 10^5 cells/ml. Enzymatic and mechanical methods were employed to break up neurospheres, respectively. Viability after dissociation was determined using the standard trypan blue exclusion test. Growth curves of NSCs dissociated by enzymatic and mechanical methods were drawn,and doubling time was calculated. To examine the effect of long-term culture on NSCs neurogenesis, neurospheres of 4,8,12,20 and 30 passages were plated on a laminin/poly-L-lysine-coated surface, and cultured for 14 days. Results In contrast with enzymatic dissociation,the method of mechanical dissociation, breaking up great neurospheres into small spheres with gradually reduced pipette, had advantages of raising cell viability and shortening cell doubling time（ P 〈 0. 05 ）. After NSCs being cultured long term in vitro, the number of neurons generated decreased with passaging, whereas the number of astrocytes increased. Conclusions The method of mechanical dissociation, helps minimize damage to NSCs and maintain the integrity of cell-cell junction and receptors of NSCs, which results in enhancing proliferation of NSCs. After expanded long term in vitro, NSCs undgo decreased neurogenesis, due to the microenviroment and inherent factors,