β淀粉样前体蛋白羧基端肽对小鼠神经母细胞瘤细胞的毒性作用  

Toxic effect of carboxyl-terminal peptide of β-amyloid precursor protein （ APPC31 ） on Neuro2a cells

作　　者：樊彩妮[1] 丁健青[1] 陈生弟[1] 汤荟冬[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院神经科,神经病学研究所,200025

出　　处：《中华神经科杂志》2010年第9期632-636,共5页Chinese Journal of Neurology

基　　金：国家重点基础研究规划“973”计划资助项目（2006cb500706）;上海自然科学基金资助项目（09JC1416402,09ZR1419100）;上海市科委重大项目资助项目（09DZ1950400）

摘　　要：目的体外探讨B淀粉样前体蛋白（amyloid precursor protein，APP）羧基端肽（APPC31）对小鼠神经母细胞瘤（Neuro2a）细胞的毒性作用及在Aβ42致毒性中的作用。方法分别将空载体质粒和过表达APPC31质粒通过脂质体转染法瞬时转染Neuro2a细胞，48h后四甲基偶氮唑盐比色（MTT）法检测细胞活力，同时利用4’，6-联脒-2-苯基吲哚（DAPI）核染色观察细胞形态，与空载体转染细胞组对照，观察APPC31对细胞有无毒性作用；然后通过相同方法，在Neur02a细胞中，分别将过表达的空载体质粒、野生型APP695质粒、APP（D664A）质粒、APP氨基端长肽（APPAC31）质粒和APPC31质粒瞬时转染Neuro2a细胞，24h后加Aβ42（10μmol／L）共孵育24h，MTT法检测细胞活力及DAPI核染色观察细胞形态，与空载体转染细胞组对照，观察Aβ42处理条件下APPC31的毒性作用；质粒转染细胞后通过免疫荧光检测目的基因表达情况。结果在无Aβ42共孵育时，空载体质粒转染组和APPC31质粒转染组细胞活力分别为：0．81±0．10、0．88±0．12，两组之间比较差异无统计学意义（t=-0．78，P=0．48），细胞核形态亦无明显凋亡或死亡改变；Aβ42（10μmol／L）共孵育条件下，无转染组、空载体质粒组、野生型APP695质粒组、APP（D664A）质粒组和APPC31质粒组细胞活力分别为：0．82±0．01、0．78±0．03、0．55±0．04、0．81±0．04、0．78±0．02、0．54±0．02，且与空载体质粒组相比，野生型APP695质粒组及APPC31质粒组细胞活力明显下降（F=47．53，P〈0．05），细胞核呈明显浓缩、染色加深改变。结论体外Neuro2a细胞单独过表达一定水平的APPC31并不能致细胞死亡；但此短肽可增强Aβ42的细胞毒性作用，为进一步明确阿尔茨海默病发病机制提供了一定的实验依据。Objective To investigate the toxic effect of the carboxyl-terminal peptide of β-amyloid precursor protein （ APPC31 ） on Neuro2a cells as well as its role in the toxic process in Neuro2a cells induced by Aβ42 in vitro. Methods The plasmid vector and the APPC31 construct were transiently transfected into Neuro2a cells respectively by lipofectamine 2000. The viability of the cells was measured by the 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay at 48 h after transfection, and their morphocytology was observed by 4＇, 6-diamidino-2-phenylindole （DAPI） nucleus staining. Afterword different constructs including vector, WTAPP695, APP（ D664A）, the amino-terminal peptide of β-amyloid precursor protein （APPAC31） and APPC31 were transiently transfected into Neuro2a cells respectively via the same method. At 24 h after transfection AI342 was added into the culture medium of Neuro2a cells with the desired concentration for another 24 h for cell studies. The viability and morphocytology of the cells were measured by using the MTT assay and DAPI nucleus staining, respectively. Results When incubated in the absence of Aβ42 , the viability of ceils transfected with vector and APPC31 construct were 0. 81 ± 0. 10 and 0. 88 ± 0. 12 respectively, and accordingly there was no significant difference between the these two groups （ t = - 0. 78, P = 0.48 ） ; meanwhile no obvious cell nuclear morphological changes of apoptosis or death occurred. However when incubated in the presence of AI342, the viability of cells transfected with vector, WTAPP695, APP（D664A） , APPAC31 and APPC31 constructs were 0. 82 ± 0. 01, 0.78 ± 0. 03,0. 55 ± 0. 04, 0. 81 ±0. 04, 0. 78 ± 0. 02 and 0. 54 ± 0. 02 respectively. The viability of cells transfected with WTAPP construct and APPC31 construct decreased significantly （ F = 47.53, P 〈 0. 05 ） compared with the control group, meanwhile cells displayed condensed nuclear and even nuclear fragmentation. Conclusions In vitro, over-expres

关 键 词：淀粉样Β蛋白前体 阿尔茨海默病 毒理学 细胞系 肿瘤 

分 类 号：R749.16[医药卫生—神经病学与精神病学]