胰岛素对阿尔茨海默病细胞模型淀粉样前体蛋白代谢的影响  

Insulin promotes amyloid precursor protein metabolism in an Alzheimer’s disease cell model

作　　者：胡容 王丹[1] 楼丹丹 李廷玉[1] 周卫辉[1] 宋伟宏 HU Rong;WANG Dan;LOU Dandan;LI Tingyu;ZHOU Weihui;SONG Weihong(Institute of Pediatrics,Key Laboratory of Child Development and Disorders of Ministry of Education,Chongqing International Science and Technology Cooperation Base for Child Development and Critical Disorders,Chongqing Key Laboratory of Pediatrics,Children's Hospital of Chongqing Medical University,Chongqing,400014,China)

机构地区：[1]重庆医科大学附属儿童医院儿科医学研究所,儿童发育疾病研究教育部重点实验室,儿童发育重大疾病国家国际科技合作基地,儿科学重庆市重点实验室,重庆400014

出　　处：《第三军医大学学报》2020年第12期1201-1208,共8页Journal of Third Military Medical University

摘　　要：目的探索胰岛素通过影响淀粉样前体蛋白(amyloid precursor protein,APP)代谢对阿尔茨海默病(Alzheimer’s disease,AD)细胞模型的作用。方法采用不同浓度的胰岛素处理人神经母细胞瘤细胞株SH-SY5Y和非神经源性细胞株HEK293、2EB2,CCK-8法检测胰岛素对细胞活性的影响;Western blot检测2EB2细胞和SH-SY5Y细胞经不同浓度胰岛素处理后APP、CTF、BACE1、ADAM10和PS1的表达,ELISA法检测Aβ40和Aβ42的水平;蛋白芯片筛选胰岛素介导的关键信号通路分子,Western blot检测上述细胞中胰岛素对信号通路AKT以及GSK3β蛋白磷酸化水平的影响。结果CCK-8检测结果显示,0~10μmol/L胰岛素浓度未对实验细胞的活性产生影响(P>0.05)。Western blot结果显示,与未处理组相比,1.00、10.00μmol/L胰岛素处理增加了APP、CTF的表达(P<0.05);在APP代谢关键酶中,BACE1表达显著增加(P<0.05)。ELISA结果显示1.00、10.00μmol/L胰岛素干预后,Aβ42的水平显著增加(P<0.05)。同时,蛋白芯片发现GSK3β为胰岛素作用的潜在靶点。Western blot结果显示,磷酸化AKT和磷酸化GSK3β蛋白的表达均下调,但差异无统计学意义(P>0.05)。结论胰岛素对阿尔茨海默病致病性产物的产生有增强作用。Objective To investigate how insulin affects the metabolism of amyloid precursor protein(APP)in a cell model of Alzheimer’s disease(AD).Methods Human neuroblastoma SH-SY5Y cells and the nonneural cell lines HEK293 and 2EB2 were treated with different concentrations of insulin for 48 h,and the changes in cell viability were evaluated with CCK-8 assay.The expression levels of APP,C-terminal fragment of APP(CTF),β-site APP cleaving enzyme 1(BACE1),theα-secretase ADAM10(a disintegrin and metalloproteinase 10)andγ-secretase presenilin 1(PS1)in the cells were detected using Western blotting.ELISA was used to determine the cellular expression levels of Aβ40 and Aβ42.The key signaling pathways mediated by insulin were screened using a protein chip.The effect of insulin on protein phosphorylation levels of AKT and GSK3βpathways was examined by detecting p-AKT and p-GSK3βexpression in the cells with Western blotting.Results Treatment with insulin below 10μmol/L did not obviously affect the viability of the cells(P>0.05).Western blotting showed that treatment with 1.00 and 10.00μmol/L insulin significantly increased the protein expression of APP and CTF(P<0.05)and also the expression of BACE1,a key enzyme for APP metabolism(P<0.05).The results of ELISA demonstrated that the expression level of Aβ42 increased significantly in the cells following treatment with 1.00 and 10.00μmol/L insulin(P<0.05).The protein microarray data identified GSK3βas a potential target of insulin.Western blot analysis showed that the expression of p-AKT and p-GSK3βproteins was down-regulated following insulin treatment,but the differences were not statistically significant(P>0.05).Conclusion Insulin promotes the metabolism of APP into CTF and Aβ42,which play certain rolesin pathogenesis of AD.

关 键 词：阿尔茨海默病 胰岛素 淀粉样前体蛋白 Β淀粉样蛋白 AKT/GSK3β 

分 类 号：R341[医药卫生—基础医学] R745.7