CAJ | 학술논문

目的：探讨锂剂对神经干细胞（neural stem cells，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Fischer 344新生大鼠侧脑室下区培养的 NSCs 为细胞模型，首先采用Cyquant DNA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S100β，行神经元标记物Tuj1细胞免疫化学染色，应用Western Blotting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GSK3β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），S100β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1mM及3mM，前者可能是通过锂剂下游经典作用通路GSK3β实现，而后者可能通过GSK3β旁路途径实现。
목적：탐토리제대신경간세포（neural stem cells，NSCs）분화적조공작용급궤제，위우화NSCs이식치료SCI 료효제공이론기출。방법：이Fischer 344신생대서측뇌실하구배양적 NSCs 위세포모형，수선채용Cyquant DNA정량법측정대조조급0.5、1、3、5mM리제처리조재분화조건하적생장곡선；동시결합성형효질세포（AST）표기물GFAP급S100β，행신경원표기물Tuj1세포면역화학염색，응용Western Blotting단백정량법검측불동농도리제대NSCs분화종산물중신경원급AST총량적영향，동시채용TUNEL조망검측법심조리제적비독성농도구간，이배제혼잡인소。최후통과여기하유경전작용통로GSK3β억제제SB216763대NSCs증식、분화、조망적작용비교，탐구리제적가능작용궤제。결과：TUNEL조망검측현시체외리제적비독성농도구간위（0～3mM）；재차범위내，1mM리제처리조중Tuj+세포총수교대조조제고약1.47±0.06배（P<0.05），SB216763처리조Tuj+세포총수제고약2.25±0.07배（P<0.05）；3mM리제처리조중GFAP+세포총수위대조조적0.55±0.02배（P<0.05），S100β여GFAP 염색결과추세일치，SB216763처리조중 GFAP+세포총수위대조조적0.90±0.06배（P>0.05）。결론：리제가명현촉진NSCs향신경원방향분화병AST적분화，기최가작용농도분별위1mM급3mM，전자가능시통과리제하유경전작용통로GSK3β실현，이후자가능통과GSK3β방로도경실현。
Objectives: To investigate the effect and its underlying mechanism of lithium on modulating neu-ral stem cells differentiation. Methods: NSCs were isolated from the subventricular 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 Tuj1 and astrocytes marker GFAP and S100 β, 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 GSK3β in-hibitor-SB216763′s effect on regulating neuraonal and astrocytes were tested and compared. Results: Lithium concentration lower than 3mM was non-toxic in differentiating NSCs. 1mM lithium stimulated neuronal pro-duction by 1.47±0.06 folds(P<0.05), while SB216763 increased by 2.25±0.07 folds(P<0.05), 3mM lithium re-duced GFAP+ cell number to 0.55 ±0.02 folds (P<0.05) compared with control group, S100β 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 1mM with its inhibition on doumregulating GSK3β pathway, mean-while suppresses astrogliogenesis at concentration of 3mM, which may be mediated by non-canonical pathway.