β淀粉样蛋白42寡聚体诱导神经元毒性和阿尔茨海默病病理形成  

Amyloidβ42oligomers induce neurotoxicity and pathogenesis of Alzheimer's disease

作　　者：邓佳君 陶倩 刘斌[1] 罗衍愈 朱仪 岳峰 Deng Jiajun;Tao Qian;Liu Bin;Luo Yanyu;Zhu Yi;Yue Feng(Hainan Provincial Key Laboratory of Biomedical Engineering,School of Biomedical Engineering,Hainan University,Haikou 570228,Hainan Province,China)

机构地区：[1]海南大学生物医学工程学院海南省生物医学工程重点实验室,海口570228 [2]昆明理工大学灵长类转化医学研究院

出　　处：《中华老年心脑血管病杂志》2024年第5期562-566,共5页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：科技创新2030重大项目(2021ZD0200903);海南省重点研发计划项目(ZDYF2021SHFZ049);海南省自然科学基金(322RC588)。

摘　　要：目的探究β淀粉样蛋白(Aβ)42寡聚体对神经元毒性的作用及诱导产生类似阿尔茨海默病(AD)病理的机制。方法采用Western blot检测和透射电子显微镜对合成的Aβ_(42)寡聚体进行鉴定。为评估Aβ_(42)寡聚体的影响,使用2,5-二苯基四氮唑溴盐检测细胞活性,将10μmol/L的Aβ_(42)寡聚体与人神经母细胞瘤系(SH-SY5Y)细胞共孵育12h和24h,与人胚胎干细胞(hESC)衍生的谷氨酸能神经元共孵育24h、48h和96h,同时设立相应对照组。原位末端标记法检测谷氨酸能神经元凋亡情况;免疫荧光染色法检测AD相关病理Aβ斑块及p-tau病理变化。结果Western blot显示,Aβ_(42)寡聚体样品中观察到单体和小分子量聚集体(<30000)。透射电子显微镜成像显示,Aβ_(42)寡聚体主要呈圆球形颗粒状。10μmol/L的Aβ_(42)寡聚体与SH-SY5Y细胞共孵育12h、24h,细胞存活率显著低于其对照组[(70.89±2.54)%vs(100.00±2.02)%,(52.63±3.37)%vs(100.00±2.80)%,P<0.05]。10μmol/L的Aβ_(42)寡聚体与谷氨酸能神经元共孵育24h、48h和96h,细胞存活率显著低于其对照组(P<0.05)。10μmol/L的Aβ_(42)寡聚体与谷氨酸能神经元共孵育48h、96h,凋亡率显著高于其对照组[(1.44±0.31)%vs(1.00±0.38)%,(1.75±0.64)%vs(1.00±0.31)%,P<0.05]。10μmol/L的Aβ_(42)寡聚体与谷氨酸能神经元共孵育24h、48h、96h,均产生呈圆形颗粒状的Aβ斑块阳性信号,其对照组均无Aβ斑块阳性信号。10μmol/L的Aβ_(42)寡聚体与谷氨酸能神经元共孵育24h,细胞在Thr231位点处产生tau过度磷酸化,Aβ_(42)寡聚体组平均荧光强度显著高于其对照组(P<0.05)。结论Aβ_(42)寡聚体对SH-SY5Y细胞和谷氨酸能神经元均具有毒性,可以诱导谷氨酸能神经元产生AD样病理。Objective To determine the neurotoxic effects of amyloid beta 42(Aβ_(42))oligomers and investigate the mechanism of their induction of Alzheimer's disease(AD)-like pathogenesis in neuronal cells.Methods Western blotting and transmission electron microscopy were used to identify the synthesized Aβ_(42)oligomers.In order to assess the impact of the oligomers,MTT assay was employed to measure cell viability in neuroblastoma cell line SH-SY5Ytreated with 10μmol/L Aβ_(42)oligomers for 12or 24h,glutamatergic neurons derived from human embryonic stem cells(hESC)exposed to Aβ_(42)oligomers for 24,48,or 96h,and corresponding control cells.TUNEL assay was utilized to assess the apoptosis of glutamatergic neurons.Additionally,immunofluorescence assay was applied to detect the changes in Aβplaques and p-tau pathology.Results Western blotting displayed monomers and small-molecule aggregation(<30000)in our synthesized Aβ_(42)oligomers,and transmission electron microscopy showed that the synthesized oligomers were mainly in a shape of spherical particles.Treatment of 10μmol/L Aβ_(42)oligomers for 12and 24hin SH-SY5Ycells significantly decreased cell viability when compared with the control cells[(70.89±2.54)%vs(100.00±2.02)%,(52.63±3.37)%vs(100.00±2.80)%,P<0.05].The 10μmol/L oligomers treatment for 24,48and 96halso decreased cell viability in glutamatergic neurons(P<0.05).The apoptotic rates was significantly higher in glutamatergic neurons after treatment for 48and 96hwhen compared to the control cells[(1.44±0.31)%vs(1.00±0.38)%,(1.75±0.64)%vs(1.00±0.31)%,P<0.05].Furthermore,circular granular Aβ-positive plaque signals were observed in the glutamatergic neurons after treated with the oligomers for 24,48,and 96h,but no such plaque signals were seen in the control cells.Additionally,glutamatergic neurons incubation with 10μmol/L oligomers for 24hresulted in tau hyperphosphorylation at the Thr231 site,with the average fluorescence intensity significantly higher than that in control cells(P<0.05).Conclusion Aβ

关 键 词：阿尔茨海默病 淀粉样β肽类 神经元 细胞凋亡 β淀粉样蛋白(Aβ)42寡聚体 

分 类 号：R749.16[医药卫生—神经病学与精神病学]