APP基因转染SK-N-SH细胞中阿尔兹海默病相关基因表达水平的变化

The change on expression level of Alzheimer disease related genes in APP transfected SK-N-SH cells

目的探讨淀粉样前体蛋白(APP)表达对AD相关基因表达水平的影响。方法构建APP基因的真核表达质粒,并转染人神经母细胞瘤细胞株SK-N-SH细胞,实时定量PCR法检测转染细胞中SIRT1、Tau、PS1、PS2及Apo E4的表达水平变化。结果成功构建了APP基因的真核表达载体,相对于空载体转染组,转染24、48 h时APP基因的过表达对SK-N-SH中SIRT1基因的表达具有明显的抑制作用(P<0.01、P<0.05);对Apo E4的表达水平有一定的上调作用(P<0.001)。转染24、48 h时,PS2基因的表达水平均有一定的上调,但均未发现差异有统计学意义(P>0.05)。转染24 h时,对PS1有上调趋势,但差异无统计学意义(P>0.05),转染48h时,则有下调作用(P<0.05)。对Tau,转染24 h时有明显的上调作用(P<0.001),但转染48 h时,则有下调趋势,差异无统计学意义(P>0.05)。相对于空载体转染组,APP基因转染组的细胞活力显著降低。结论 APP基因的过表达对SK-N-SH细胞中SIRT1基因的表达具有明显的抑制作用;对Apo E4、PS2的表达水平有一定的上调作用,对Tau、PS1的表达水平有上调和下调作用不一,表明这些基因之间存在密切关系和对AD病理机制影响的复杂性,这为揭示不同AD相关基因之间的相互影响及调控机制提供一定的实验证据。

Objective To explore effects of amyloid precursor protein（APP） over-expression on gene expression of other Alzheimer disease（AD） related genes in SK-N-SH cells. Methods The recombinant eukaryotic expression vector of APP was transfected into SK-N-SH cells mediated by LipofectamineTM2000. After transtection, cells were observed by fluorescence microscope.Moreover, gene expression level of target gene of SIRT1, Tau, Apo E, PS1 and PS2 were analyzed in cells which over-expressed related gene by real-time PCR respectively. Results Eukaryotic expression vectors of APP gene were successfully constructed. Gene expression level of SIRT1 gene was down-regulated in cells which was over-expressed APP（P〈0.01, P〈0.05）. However, gene expression level of Apo E4 gene was upregulated in cells which was overexpressed APP gene（P〈0.001）. After treated either 24 h or 48 h in cells which was over-expressed APP, there was no significant up-regulation of PS2 gene expression level（P〉0.05）. After treated48 h in cells overexpressed APP gene, gene expression level of PS1 was decreased significantly（P〈0.05）. However, there was no increase of PS1 after treated 24 h in cells overexpressed APP gene. For Tau gene, gene expression level was up-regulated after treated 24 h（P0.001）, but down-regulated after treated 48 h（P〉0.05） in cells which was over-expressed APP. There was significant cytoxicity after treated 24 h or 48 h by vector, recombinant plasmid or transfection reagent. And the cell viability of SK-N-SH cells was decreased significantly（P〈0.001）. Besides, their cytoxicity was positive correlation with the fragment size which was subcloned into vector. Compared with the group treated by control vector, the cell viability of APP gene transfected SK-N-SH cells was decreased significantly（P〈0.001）. Conclusion Overexpression of APP gene show significant downregulation effects on the expressionof SIRT1 gene; but show upregulation effects on the gene expression of Apo E4 and PS2. Besides, there is different effects on Tau and PS1 genes. Results show that there are some relations between these AD-related genes and the complexity of AD pathogenic mechanism. The above results provide experimental evidence for revealing certain mutual influence between different AD-related genes and regulation mechanism.