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目的探讨氯化锂对缺氧后神经干细胞磷酸化GSK-3β蛋白表达的影响。方法提取新生SD乳鼠NSCs,接种至无糖DMEM培养基,放入95%N2+5%CO2条件下制作缺氧NSCs模型。正常组NSCs加无血清培养基,缺氧NSCs分为单纯缺氧组、生理盐水干预组、1m M、3m M、5m M氯化锂干预组;单纯缺氧组仅缺氧,生理盐水干预组缺氧后加生理盐水,3组氯化锂干预组分别加入100m M氯化锂至所需的浓度,继续在无血清培养基中培养。免疫组织化学技术检测各组GSK-3β/P-GSK-3β阳性表达细胞。结果缺氧后NSCs形态不规则,数量减少,折光性减弱,胞体肿胀,甚至出现细胞膜破裂;缺氧组死亡NSCs数(9.400±1.304)明显高于正常组(2.400±0.548)(P﹤0.05),缺氧组NSCs细胞活力(0.409±0.002)明显低于正常组(0.482±0.004)(P﹤0.05)。1m M氯化锂干预组P-GSK-3β表达较生理盐水干预组、单纯缺氧组及5m M氯化锂干预组强,较3m M氯化锂干预组弱。3m M氯化锂干预组P-GSK-3β表达较1m M、5m M氯化锂干预组强。3m M氯化锂干预组P-GSK-3β阳性细胞数较单纯缺氧组、生理盐水干预组、1m M、5m M氯化锂干预组明显增多(P﹤0.05);生理盐水干预组P-GSK-3β阳性细胞数与单纯缺氧组比较差异无统计学意义(P>0.05)。结论氯化锂可能通过PI3K/Akt/GSK-3β信号通路调控缺氧NSCs增殖。
Objective To investigate the effect of lithium chloride on phosphorylated GSK-3β protein expression in neural stem cells after hypoxia. Methods Newborn SD rat neonatal NSCs were isolated and inoculated into glucose-free DMEM medium to produce hypoxic NSCs model with 95% N2 + 5% CO2. NSCs in normal group and serum-free medium, hypoxic NSCs were divided into simple hypoxic group, saline intervention group, 1m M, 3m M, 5m M lithium chloride intervention group; hypoxia group, After hypoxia and saline, three groups of lithium chloride intervention group were added to 100m M lithium chloride to the desired concentration, continue to culture in serum-free medium. Immunohistochemistry was used to detect the expression of GSK-3β / P-GSK-3β in each group. Results The number of NSCs in hypoxic group was irregular, the number decreased, the refraction decreased, the cell body swollen, and even the ruptured cell membrane. The number of NSCs died in hypoxia group was significantly higher than that in normal group (2.400 ± 0.548 vs 2.400 ± 0.548, P <0.05) The viability of NSCs in hypoxia group (0.409 ± 0.002) was significantly lower than that in normal group (0.482 ± 0.004) (P <0.05). The expression of P-GSK-3βin 1m M lithium chloride intervention group was stronger than that in saline intervention group, simple hypoxia group and 5m M lithium chloride treatment group, but weaker than 3m M lithium chloride intervention group. The expression of P-GSK-3β in 3m M lithium chloride intervention group was stronger than 1m M, 5m M lithium chloride intervention group. The number of P-GSK-3βpositive cells in the intervention group was significantly higher than that in the hypoxia group, the saline intervention group, the 1m M, 5m M lithium chloride intervention group (P <0.05) The number of GSK-3βpositive cells was not significantly different from that of hypoxia alone group (P> 0.05). Conclusion Lithium chloride may regulate the proliferation of hypoxic NSCs through the PI3K / Akt / GSK-3β signaling pathway.