EGFP示踪NSCs海马移植对APP/PS1转基因鼠认知功能及基底前脑胆碱能神经元的影响

Effects of EGFP-traced NSCs hippocampal transplantation on cognitive function and basal forebrain cholinergic neurons in APP/PS1 transgenic mice

目的观察神经干细胞(NSCs)移植对APP/PS1双转基因小鼠认知功能及基底前脑胆碱能神经元的影响,并探讨其可能机制。方法体外分离培养EGFP转基因小鼠胎脑来源NSCs,24只12月龄雄性APP/PS1双转基因小鼠随机分入实验组(Tg-NSCs组)和AD对照组(Tg-AD组),每组12只,12只同月龄雄性野生型小鼠作为正常对照组(WT组),Tg-NSCs组进行EGFP示踪NSCs移植,其余两组进行等量磷酸盐缓冲液注射,移植部位为双侧海马区。移植4周后采用Morris水迷宫检测三组小鼠学习记忆功能,免疫荧光组化法和Western blot检测基底前脑胆碱能神经元的变化情况;Q-PCR检测海马神经营养因子NGF、BDNF、NT3的mRNA水平的变化。结果①体外悬浮培养的神经球表达EGFP阳性,免疫荧光检测显示神经干细胞特异性标志物Nestin阳性。移植4周后,可见EGFP阳性NSCs在海马注射移植部位存活并向胼胝体,海马深部和齿状回迁移,基底前脑未见EGFP阳性细胞。②与Tg-AD组相比,Tg-NSCs组基底前脑ChAT神经元及蛋白水平均增加(P<0.05),但ChAT蛋白表达低于WT组水平(P<0.05)。③Tg-NSCs组海马区神经营养因子NGF、BDNF、NT3的mRNA表达增加,高于Tg-AD组(P<0.05),且与WT组相比差异无统计学意义(P>0.05)。④水迷宫结果显示Tg-NSCs组学习记忆功能改善优于Tg-AD组(P<0.05),但仍低于WT组水平(P<0.05)。结论 NSCs海马移植并不能对该转基因鼠基底前脑胆碱能神经元丢失产生直接的替代与补充,可能通过分泌神经营养因子对基底前脑胆碱能神经元起到保护作用,从而促进其认知功能的恢复。

Objective To observe the effects of neural stem cells(NSCs)transplantation on cognitive function and basal forebrain cholinergic neurons in APP/PS1 double transgenic mice and explore the possible mechanisms. Methods Isolated and cultured NSCs derived from embryonic fetal brain of EGFP transgenic mouse in vitro. A total of 24 12-month old male APP/PS1 double transgenic AD mice were randomly divided into Tg-NSCs group and Tg-AD group,and the same age of 12 male wild type mice as a normal control group(WT group). The Tg-NSCs group received NSCs labeled by EGFP,and other two groups were injected same volume of phosphate buffer respectively. The transplant sites were bilateral hippocampus. Four weeks after transplantation,Morris water maze was used to detect the learning and memory function of the three groups of mice. The changes of cholinergic neurons and ChAT protein in the basal forebrain were detected by fluorescence immunohistochemistry and Western blot respectively. Relative mRNA levels of NGF,NT3 and BDNF in the hippocampus were detected by Q-PCR. Results ①Neurosphere cultured in vitro showed positive expression of EGFP and Nestin as neural stem cell specific marker. Four weeks after transplantation,EGFP-positive NSCs survived in the hippocampus and migrated to the corpus callosum,deep hippocampus and dentate gyrus. While no EGFP-positive cells were found in the basal forebrain.②Compared with Tg-AD group,ChAT neurons and protein levels in the basal forebrain of the Tg-NSCs group were increased(P<0.05),but the protein level of ChAT was lower than WT group(P<0.05).③The mRNA expressions of neurotrophic factors NGF,BDNF and NT3 were increased in the hippocampus of the Tg-NSCs group than those in the Tg-AD group(P<0.05),and there was no significant difference compared with WT group(P>0.05).④The results of Morris water maze showed that the improvement of learning and memory function of Tg-NSCs group was better than that of Tg-AD group(P<0.05),but it was still lower than that in WT group(P<0 05). Conclusion NSCs hippocampal transplantation could not directly replace and supplement the loss of cholinergic neurons in basal forebrain of AD mice. It may play a protective role in basal forebrain cholinergic neurons by secreting neurotrophic factors,thus alleviate cognitive impairment in AD mice.