转染早老素1突变基因SH-SY5Y细胞凋亡的研究Study on the Role of Mutant PS-1 Gene in Alzheimer Disease
冯颖,许贤豪,彭丹涛,张华,乔立艳,张星虎,刘广志,殷剑,国红,王红,李宝林,莫培生
摘要(Abstract)：
目的 探索转染早老素 -突变基因导致 SH-SY5 Y细胞凋亡的机制。方法 用 Xba I和 Acc6 5 I内切酶将携带野生型和突变型 PS-1的 p Bluescript质粒双酶切 ,电泳后纯化其 c DNA片段 ,然后将其克隆到 p CDNA3载体中。由脂质体介导将 p CDNA3空载体、携带有野生型和突变型 PS-1基因的 p CDNA3质粒分别转染到神经母细胞瘤细胞系 SH-SY5 Y细胞中。用蛋白印迹 ( Western blot)杂交检测其表达。用流式细胞仪 ( Coulter EpicsXL)检测在不同情况下不同细胞凋亡的百分率 ,荧光显微镜下观察碘化吡啶 ( PI)、Hoechst3334 2 ( Ho)双染的凋亡细胞的形态 ,并用分子生物学方法检测凋亡细胞的 DNA状况。结果 在无血清培养 2 4 h条件下含有空载体、野生型和突变型 ( A2 4 6 E) PS-1基因的 SY5 Y细胞凋亡百分率依次为 ( 6 .0 3± 2 .0 3)、( 8.34± 2 .4 1 )、( 2 6 .2±4 .6 )。用 SY5 Y细胞作为对照 ,其凋亡百分率为 ( 4 .2 2± 1 .89)。在 Aβ2 5 -35 ( 2 5 μmol/L)诱导下其凋亡百分率依次为 ( 2 4 .4± 3.6 )、( 2 7.7± 2 .5 )、( 5 0 .3± 8.3)。SY5 Y对照组为 ( 1 8.4± 3.1 )。比较显示含突变早老素 1基因细胞的凋亡程度明显升高 ( P<0 .0 0 1 )。结论 早老素 1基因突变导致神经细胞更容易在各种刺激下发生凋亡 ,
关键词(KeyWords)： Alzheimer病;早老素;突变;凋亡
基金项目(Foundation): “九五”国家医学攻关课题基金资助项目 ( 96 -90 6 -0 5 -0 7)
作者(Author): 冯颖,许贤豪,彭丹涛,张华,乔立艳,张星虎,刘广志,殷剑,国红,王红,李宝林,莫培生
Email:
参考文献(References)：
- [1]GamesD,AdamsD,AlessandrinlT,etal.Alzheimer-type neuropathology in transgenic miceoverexpressingV717Fβ-amyloid precursor protein[J].Nature,1995,373:523-527.
- [2]MarkRJ,LovellMA,MarkesberyWR,etal.A rolefor4-hydroxynonenal in disruption of ion homeostasisand neuronal death induced by amyloidβ-peptide[J].JNeurochem,1997,68:255-264.
- [3]DoanA,ThinalaranG,BorcheltDR,et al.Proteintopology of presenilin-1[J].Neuron,1996,17:1023-1030.
- [4]GschwindM,HuberG.Apoptotic cell death inducedbyβ-amyloid1-42 peptide iscell type-dependent[J].JNeurochem,1995,65:292-300.
- [5]RukensteinA,RydelRE,GreeneLA.Multiple agentsrescuePC12 cells from serum-free cell death by trans-lation-and transcription-independentmechanisms[J].JNeurosci,1991,11:2552-2563.
- [6]LooDT,CopaniA,PikeCJ,etal.Apoptosis isinduced byβ-amyloid in cultured central nervous sys-tem neurons[J].ProcNatlAcadSciUSA,1993,90:7951-7955.
- [7]PrehnJH,BindokasVP,MareuccilliCJ,etal.Regula-tion of neuronalBcl2 protein expression and calciumhomeostasis by transforming growth factor type betaconfers wideranging protection on rat hippocampalneurons[J].ProcNatlAcadSciUSA,1994,91:12599-12603.
- [8]GuoQ,SopherBL,FurukawaK,et al.Alzheimer spresenilin mutation sensitizes neural cells to apoptosisinduced by trophic factor withdrawal and amyloidβ-peptide:involvementof calcium and oxyradicals[J].JNeurosci,1997,17(11):4212-4222.
- [9]LamM,DubyakG,DistelhorstCW.Effect of gluco-corticosteroid treatment on intracellular calciumhomeostasis in mouse lymphoma cells[J].MolEndocrinol,1993,7:686-693.
- [10]WolozinB,IwasakiK,VitoP,etal.Participation ofpresenilin2 in apoptosis:enhanced basal activity con-ferred by anAlzheimer mutation[J].Science,1996,274:1710-1713.
- [11]QuerfurthHW,SelkoeDJ.Calcium ionophoreincreases amyloidβ-peptide production by culturedcells[J].Biochemistry,1994,33:4550-4561.
- [12]HuttonM,HardyJ.The presenilins andAlzheimer sdisease[J].Hum.MolecGenet,1997,6:1639-1646.
- [13]WolozinB,AlexanderP,PalacinoJ.Regulation ofapoptosis by presenilin1[J].NeurobiolAging,1998,19( suppl1):S23-S27.
- [14]GuoQ,FurukawaK,SopherBL,et al.Alzheimer sPS-1 mutation perturbs calcium homeostasisand sen-sitizesPC12 cells to death induced by amyloidβ-pep-tide[J].NeuroReport,1996,8:379-383.
- [15]TaniiH,AnkarcronaM,FloodF,et al.Alzheimer'sdisease presenilin-1Exon9deletion andL250S muta-tions sensitizeSH-SY5Y neuroblastoma cells tohyperosmotic stress-induced apoptosis[J].Neuro-science,2000,95:593-601