神经干细胞联合神经生长因子纳米粒海马移植对阿尔茨海默病转基因鼠的影响

Effects of Neural Stem Cells combine with Nerve Growth Factor Nanoparticles Transplantation in Alzheimer's Disease Transgenic Mice

目的:观察神经生长因子(NGF)纳米粒对神经干细胞(NSC)在APP/PS1转基因鼠脑内存活、迁移、分化的影响,以及NSC联合NGF纳米粒移植对基底前脑胆碱能神经元及海马、皮层β-淀粉样蛋白(Aβ)的影响。方法:36只12月龄雄性APP/PS1转基因小鼠采用随机数字表法分为AD对照组、NSC移植组、联合移植组,每组12只。另外选取12只12月龄野生型(WT)小鼠作为WT对照组。体外分离培养表达增强型绿色荧光蛋白(EGFP)转基因小鼠胎脑来源的NSC。WT对照组和AD对照组于双侧海马分别注射5μL磷酸盐缓冲液(PBS),NSC移植组和联合移植组于双侧海马分别注射等量NSC悬液和NSC联合NGF纳米粒悬液。移植4周后,采用免疫荧光法检测移植NSC的存活、迁移、分化以及基底前脑胆碱能神经元(ChAT)的变化;采用实时荧光定量PCR法(Q-PCR)检测基底前脑ChAT和Lhx8 mRNA表达;采用Western blotting检测各组基底前脑ChAT蛋白表达情况;采用硫磺素T染色检测各组小鼠海马及皮层Aβ斑块的数量。结果:(1)移植4周后,EGFP阳性NSC在脑内存活,广泛迁移至胼胝体远端、皮层及海马深部,并能分化为神经元及星形胶质细胞,联合移植组细胞存活数量更多,并且NSC显示更多复杂的突起形态。(2) NSC移植组和联合移植组基底前脑ChAT神经元数量增加(P<0.05),联合移植组ChAT及Lhx8基因表达明显高于NSC移植组(P<0.05)。(3) NSC移植组和联合移植组海马及皮层Aβ斑块的数量均未明显减少(P>0.05)。结论:NSC联合NGF纳米粒移植可以间接保护基底前脑胆碱能神经元和NSC的存活与成熟,并促进基底前脑ChAT和Lhx8的基因表达,但不能减少海马和皮层中Aβ斑块数量。

Objective:To observe the effect of nerve growth factor(NGF)nanoparticles on the survival,migration and differentiation of transplanted neural stem cells(NSC)in the brain of APP/PS1 transgenic mice,and the effect of NSC combined with NGF nanoparticles transplantation on cholinergic neurons in basal forebrain and amyloidβ-protein(Aβ)in the hippocampus and cortex.Methods:A total of 3612-month-old male APP/PS1 transgenic mice were randomly divided into the AD control group,the NSC transplant group and the combined group,with 12 cases in each group.A total of 12-month-old wild type(WT)mice were selected as the WT control group.NSC derived from fetal brain of transgenic mice expressing enhanced green fluorescent protein(EGFP)was isolated and cultured in vitro.5μL phosphate buffer(PBS)were injected in bilateral hippocampus of the WT control group and the AD control group,5μL NSC suspension and NSC combined with NGF nanoparticle suspension were injected in the bilateral hippocampus of the NSC transplant group and the combined group respectively.After transplantation for four weeks,immunofluorescence method was used to detect the survival,migration,differentiation of transplanted NSC and the change of ChAT in the basal forebrain.Real-time quantitative PCR(Q-PCR)was used to detect the mRNA expressions of ChAT and Lhx8 in the basal forebrain.Western blotting was used to detect ChAT protein expression in the basal forebrain.Thioflavine-T staining was used to detect the numbers of Aβplaques in the hippocampus and cortex.Results:①After transplantation for four weeks,EGFP-positive NSC survived in the brain,migrated extensively to corpus callosum,cortex and deep hippocampus,and differentiated into neurons and glial cells,NSC in the combined group survived more than those in the NSC transplant group and showed more complex synaptic morphology.②The number of ChAT neurons in the basal forebrain of the NSC transplant group and the combined group increased significantly(P<0.05),compared with the NSC transplant group,the gene expression of ChAT and Lhx8 in the combined group were increased significantly(P<0.05).③The number of Aβplaques in hippocampus and cortex of the NSC transplant group and the combined group didn't decreased significantly(P>0.05).Conclusion:NSC combined with NGF nanoparticles transplantation could indirectly protect cholinergic neurons in the basal forebrain and the survival and maturation of transplanted NSC,and promote the gene expression of ChAT and Lhx8 in the basal forebrain,but could not reduce the number of Aβplaques in the hippocampus and cortex.