稳定表达人野生型淀粉样前体蛋白细胞模型的建立与鉴定  

Establishment and identification of cell lines stable-expressing human wild amyloid precursor protein

作　　者：王芬[1] 方伯言[2] 贾建平[1] 

机构地区：[1]首都医科大学宣武医院神经内科,北京100053 [2]辽宁医学院附属第一医院神经内科

出　　处：《中国现代神经疾病杂志》2009年第3期275-279,共5页Chinese Journal of Contemporary Neurology and Neurosurgery

基　　金：国家重点基础研究发展计划(973计划)项目(项目编号:2006cb500700);北京市自然科学基金资助项目(项目编号:7071004)

摘　　要：目的建立和鉴定稳定表达人野生型淀粉样前体蛋白（APP）的人神经母细胞瘤细胞系SH—SY5Y细胞模型。方法脂质体转染法将含人野生型APP751cDNA的真核表达质粒pcDNA3／APPAP751^WT转染至SH—SY5Y细胞，G418对转染后细胞进行筛选，获得稳定表达人野生型APP751，细胞模型。Western blotting法、MTT比色法和放射免疫法检测细胞APP表达水平，细胞生长情况和细胞内外β-淀粉样蛋白（Aβ）表达水平。结果转染组细胞APP表达水平明显高于未转染组和空载体组。未转染组和空载体组细胞滞留期为12h，对数生长期为12h-4d；转染组细胞滞留期为24h，对数生长期为1～4d；培养12h后，转染组细胞活力低于未转染组和空载体组（t=4．363，P=0.002；t=4．040，P=0．003），培养1d后，转染组细胞活力降低，且低于未转染组和空载体组（t=4．736，P=0．001；t=4．317，P=0．005），其余各时间点差异均无统计学意义（P〉0．05）。转染组细胞细胞外AB表达水平高于未转染组和空载体组（t=7．690；P=0．000；t=7．448，P=0．000），而3组细胞细胞内Aβ表达水平则差异无统计学意义（P〉0.05）。结论成功建立稳定表达人野生型APP751的人神经母细胞瘤细胞系SH-SY5Y细胞模型，为进一步研究阿尔茨海默病的发病机制提供了良好的细胞模型。Objective To establish and identify the human neuroblastoma cell lines SH-SY5Y stable＇ expressing human wild amyloid precursor protein （APP）. Methods The pcDNA3/APP APP751^WT containing human wild APP751 cDNA was transfected into SH-SY5Y cells by Lipofectamine, and stably transfected cell clones were screened by G418. The expression of APP was detected with polyclonal rabbit anti-human APP C terminal antibody and monoclonal mouse anti-human Aβ1-17 antibody by Western blotting, the cells growth was assessed by methyl＇thiazol-tetrazolium （MTT） assay, and the expressions of intracellular and extracellular β- amyloid （Aβ） were detected by radioimmunity assay. Resets Western blotting confirmed that the expression of APP was significantly higher in transfected group than that in normal and mock transfected groups. The cells retention period and logarithmic phase were 12 h and 12 h-4 d, respectively, in normal and mock tranfected groups, and were 24 h and 1-4 d, respectively, in transfected group. After 12 h-culture, the viability of cells in transfected group was lower than that in normal and mock transfected groups （t = 4.363, P= 0.002; t = 4.040, P = 0.003）. After 1 d-culture, the cells viability in transfected group reduced and was lower than that in normal and mock transfected groups （t = 4.736, P= 0.001; t = 4.317, P= 0;005）, but no significant differences were seen at other time points （P〉 0.05, for all）. The expression of extracellular Aβ in cells of transfected group was significantly higher than that in cells of normal and mock transfected groUps （t = 7.690, P= 0.000; t = 7.448, P= 0.000）, but the difference of the expression of intracellular Aβ in cells of these 3 groups was not significant （P〉 0.05）. Conclusion Human neuroblastoma cell lines SH-SY5Y stable-expressing human wild APP751 is successfully established, which may provide a good celt model to explore the pathogenesis of Alzheimer disease （AD）.

关 键 词：阿尔茨海默病 淀粉样Β蛋白前体 神经母细胞瘤 细胞系 肿瘤 转染 

分 类 号：R749.16[医药卫生—神经病学与精神病学] R576[医药卫生—临床医学]