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浙江大学学报（医学版）
浙江大学学报（医学版）  2018, Vol. 47 Issue (5): 473-479    DOI: 10.3785/j.issn.1008-9292.2018.10.05
专题报道     
蛇床子素通过上调微RNA-101a-3p抑制阿尔茨海默病细胞淀粉样前体蛋白表达
蔺莹(),姚璎珈,梁喜才,时悦,孔亮,肖洪贺,吴雨桐,倪颖男,杨静娴*()
辽宁中医药大学药学院, 辽宁 大连 116600
Osthole suppresses amyloid precursor protein expression by up-regulating miRNA-101a-3p in Alzheimer's disease cell model
LIN Ying(),YAO Yingjia,LIANG Xicai,SHI Yue,KONG Liang,XIAO Honghe,WU Yutong,NI Yingnan,YANG Jingxian*()
College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, Liaoning Province, China
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摘要：

目的: 研究阿尔茨海默病细胞模型中蛇床子素抑制淀粉样前体蛋白（APP）的作用及机制。方法: 脂质体2000转染建立APP高表达的SH-SY5Y细胞模型。利用MTT和乳酸脱氢酶（LDH）法检测蛇床子素对APP高表达细胞的存活率和损伤程度的影响。利用基因芯片技术筛选给予蛇床子素治疗后差异表达的微RNA（miRNA），然后通过生物信息学分析预测与差异表达miRNA靶向结合的基因。细胞内转入差异表达miRNA的抑制剂，然后采用免疫荧光细胞化学法观察细胞中APP、β淀粉样蛋白（Aβ）表达情况，RT-PCR法检测细胞中APP mRNA表达。结果: 实验成功构建了APP高表达的阿尔茨海默病细胞模型。MTT和LDH法检测结果显示，蛇床子素对于APP高表达的细胞具有一定的保护作用，可以减轻细胞的损伤。miRNA-101a-3p是蛇床子素调控较明显的miRNA，其与APP的3'非翻译区靶向结合。与模型对照组比较，miR-101a-3p抑制剂组APP和Aβ蛋白荧光强度增加，APP mRNA表达量增加（均P < 0.01）；加入蛇床子素后细胞中APP和Aβ蛋白荧光强度降低，APP mRNA表达量减少（均P < 0.01）。结论: 在阿尔茨海默病细胞模型中，蛇床子素通过上调miR-101a-3p抑制APP表达。
关键词： 阿尔茨海默病;  蛇床子素/药理学;  微RNAs;  淀粉样β蛋白前体;  蛋白质阵列分析;  芯片分析技术    
Abstract:

Objective: To investigate the effect of osthole on the expression of amyloid precursor protein (APP) in Alzheimer's disease (AD) cell model and its mechanism. Methods: The SH-SY5Y cell with over expression of APP was established by transfection by liposome 2000. The cells were treated with different concentrations of osthole, and the cell viability was determined by MTT and lactate dehydrogenase (LDH) assay. The differentially expressed miRNAs with and without osthole treatment were detected by miRNA array, and the target genes binding to the differentially expressed miRNAs were identified and verified by databases and Cytoscape. After the inhibitor of the differentially expressed miRNA was transduced into cells, the changes of APP and amyloid β (Aβ) protein were determined by immunofluorescence cytochemistry, and the mRNA expression of APP was determined by RT-PCR. Results: The AD cell model with over expression of APP was established successfully. The results of MTT and LDH assay showed that osthole had a protective effect on cells and alleviated cell damage. miR-101a-3p was identified as the differentially expressed miRNA, which was binding to the 3'-UTR of APP. Compared with APP group, the expression of APP and Aβ protein and APP mRNA increased in the miR-101a-3p inhibitor group (all P < 0.01), while the expression of APP and Aβ protein and APP mRNA decreased in the cells with osthole treatment (all P < 0.01). Conclusion: Osthole inhibits the expression of APP by up-regulating miR-101a-3p in AD cell model.
Key words: Alzheimer disease    Osthole/pharmacology    MicroRNAs    Amyloid beta-protein precursor    Protein array analysis    Microchip analytical procedures
收稿日期: 2018-07-05 出版日期: 2019-01-23
:  R742  
基金资助: 国家自然科学基金(81173580)
通讯作者: 杨静娴     E-mail: lnutcm_ly@163.com;jingxianyang@yahoo.com
作者简介: 蔺莹(1993-), 女, 硕士研究生, 主要从事神经药理学研究; E-mail:lnutcm_ly@163.com; https://orcid.org/0000-0003-1809-1839
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蔺莹
姚璎珈
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时悦
孔亮
肖洪贺
吴雨桐
倪颖男
杨静娴

引用本文:

蔺莹,姚璎珈,梁喜才,时悦,孔亮,肖洪贺,吴雨桐,倪颖男,杨静娴. 蛇床子素通过上调微RNA-101a-3p抑制阿尔茨海默病细胞淀粉样前体蛋白表达[J]. 浙江大学学报（医学版）, 2018, 47(5): 473-479.

LIN Ying,YAO Yingjia,LIANG Xicai,SHI Yue,KONG Liang,XIAO Honghe,WU Yutong,NI Yingnan,YANG Jingxian. Osthole suppresses amyloid precursor protein expression by up-regulating miRNA-101a-3p in Alzheimer's disease cell model. J Zhejiang Univ (Med Sci), 2018, 47(5): 473-479.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2018.10.05    或    http://www.zjujournals.com/med/CN/Y2018/V47/I5/473

图 1  APP、GFP转染人神经母细胞瘤细胞的绿色荧光图
图 2  各组细胞存活率比较(n=3)
图 3  微RNA在各组间差异表达聚类热图
图 4  免疫荧光细胞化学法观察APP和Aβ蛋白表达
图 5  各组APP和Aβ蛋白表达量比较
图 6  各组miRNA-101a-3p和APP mRNA表达及比较
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