摘要
目的 :观察 β 淀粉样前体蛋白第二、三外显子编码结构域对培养的人神经纤维母细胞瘤细胞系 (SH SY5Y)生长代谢和 β淀粉样蛋白 (Aβ)分泌的影响。 方法 :利用重组DNA技术 ,构建 pcDNA3.1APP695和删除第二、三外显子的pcDNA3.1ΔAPP695质粒 ,用脂质体转染法将其转入SH SY5Y细胞系 ,用 0 .6 g·L-1G4 18筛选获得稳定表达外源基因的细胞系 ,采用MTT法分析对细胞增殖的影响 ,用免疫沉淀Westernblot分析细胞培养液中Aβ的含量。结果 :成功构建了APP695cDNA和删除第二、三外显子的ΔAPP695cDNA的真核表达载体 ,转染SH SY5Y细胞系后 ,Westernblot证实外源基因得到表达 ,MTT分析结果显示删除第二、三外显子的ΔAPP695cDNA未对细胞产生明显的抑制增殖作用 ,免疫沉淀Westernblot分析表明 ,与转染全长APP695cDNA细胞相比 ,转染ΔAPP695cDNA细胞Aβ分泌显著减少。 结论 :APP695第二、三外显子编码结构域删除后抑制了Aβ的生成 。
Objective: To explore the effect of Aβ secretion lacking exons 2 and 3 of APP 695 (ΔAPP 695 ) on SH SY5Y neuroblastoma cells. Methods: APP 695 cDNA and ΔAPP 695 cDNA eukaryotic expression vectors were constructed by gene recombination. Immunoprecipitation Western blotting was used to analyse the Aβ secretion and MTT assay to observe the cell viability's change after being stably transfected with the plasmids by lipofectamine method. Results: Stable transfectants were selected in 0.6 g·L -1 G418. ΔAPP 695 cDNA construct transfection decreased significantly the production of β amyloid in the SH SY5Y compared with APP 695 cDNA construct. It did not affect the cell viability by MTT method. Conclusion: This result suggests that the alternative splicing of APP exons 2 and 3 modulates the secretion of Aβ by influencing the processing of APP.
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2003年第3期261-265,共5页
Journal of Peking University:Health Sciences
基金
国家自然科学基金 ( 3 9870 2 76
3 9840 0 10 )
国家重点基础研究发展规划项目基金 (G19990 64 0 0 7)~~
