摘要
目的:探讨锂剂对神经干细胞(neuralstemcells,NSCs)分化的调控作用及机制,为优化NSCs移植治疗SCI疗效提供理论基础。方法:以Fischer344新生大鼠侧脑室下区培养的NSCs为细胞模型,首先采用CyquautDNA定量法测定对照组及0.5、1、3、5mM锂剂处理组在分化条件下的生长曲线;同时结合星形胶质细胞(AST)标记物GFAP及$10013,行神经元标记物Tujl细胞免疫化学染色.应用WesternBlotting蛋白定量法检测不同浓度锂剂对NSCs分化终产物中神经元及AST总量的影响,同时采用TUNEL凋亡检测法寻找锂剂的非毒性浓度区间,以排除混杂因素。最后通过与其下游经典作用通路GSK3[3抑制剂SB216763对NSCs增殖、分化、凋亡的作用比较,探究锂剂的可能作用机制。结果:TUNEL凋亡检测显示体外锂剂的非毒性浓度区间为(0。3mM):在此范围内.1mM锂剂处理组中Tuj+细胞总数较对照组提高约1.47+0.06倍(P〈0.05),SB216763处理组Tuj+细胞总数提高约2.25+0.07倍(P〈0.05);3mM锂剂处理组中GFAP-细胞总数为对照组的0.55+0.02倍(P〈0.05),S100[3与GFAP染色结果趋势一致,SB216763处理组中GFAP-细胞总数为对照组的0.90+0.06倍(P〉0.05)。结论:锂剂可明显促进NSCs向神经元方向分化并AST的分化,其最佳作用浓度分别为lmM及3mM.前者可能是通过锂剂下游经典作用通路GSK3[3实现,而后者可能通过GSK3[3旁路途径实现。
Objectives: To investigate the effect and its underlying mechanism of lithium on modulating neu- ral stem cells differentiation. Methods: NSCs were isolated from the subventrieular zone of neonatal Fischer 344 Rat. The growth curve of differentiating NSCs was detected by using Cyquant assay. Immunocytochemistry staining combining with BrdU incorporation assay were used to detectneuronal marker Tujl and astrocytes marker GFAP and $100(3, the percentage, as well as the total numbers of each lineage were also estimated. TNUEL apoptosis assay was also performed to rule out the lithium toxicity. Finally, lithium and GSK3[3 in- hibitor-SB216763's effect on regulating neuraonal and astroeytes were tested and compared. Results: Lithium concentration lower than 3mM was non-toxic in differentiating NSCs. lmM lithium stimulated neuronal pro- duction by 1.47_+0.06 folds(P〈0.05), while SB216763 increased by 2.25±0.07 folds(P〈O.05), 3mM lithium re- duced GFAP+ cell number to 0.55+0.02 folds (P〈0.05) compared with control group, S10013 staning result showed the similar tendency, conversely, SB216763 did not reduce GFAP+ cell number, with 0.90±0.06 folds compared with control group (P〉0.05). Conclusions: Lithium exerts dual roles on NSCs's differentiation. It stimulates neurogenesis at concentration of lmM with its inhibition on doumregulating GSK3β pathway, mean- while suppresses astrogliogenesis at concentration of 3raM, which may be mediated by non-canonical pathway.
出处
《中国脊柱脊髓杂志》
CAS
CSCD
北大核心
2013年第12期1101-1108,共8页
Chinese Journal of Spine and Spinal Cord
基金
国家自然科学基金(项目编号:81200944/H0910)
中国博士后基金(项目编号:2012M520044)
关键词
锂剂
神经干细胞
分化调控
Lithium
Neural stem cells
Differentiation