禹文峰,孔欣,官志忠
YU Wenfeng,KONG Xin,GUAN Zhizhong

• 1:贵州医科大学分子生物学重点实验室
• 2:贵州医科大学病理学教研室

摘要(Abstract)：

目的:改进Aβ_(1-42)寡聚体的制备与鉴定方式。方法:以化学合成多肽Aβ_(1-42)为原料在体外制备Aβ_(1-42)寡聚体,用Western blotting进行鉴定;加入不同浓度Aβ_(1-42)寡聚体后,用CCK-8法测定体外原代培养大鼠大脑皮质星形胶质细胞(AS)的存活率。结果:化学合成的Aβ_(1-42)寡聚体分子量为4~188 k D,以64 k D最多,亦有少量4 k D单体存在;CCK-8法显示Aβ_(1-42)寡聚体对AS有明显的神经毒性作用,并呈现剂量-效应关系。结论:成功建立了一种改进的制备和鉴定Aβ_(1-42)寡聚体的方法,为深入研究阿尔茨海默病提供了研究手段。
Objective: To improve method for preparation and identification of Aβ_(1-42) oligomer.Methods: Aβ_(1-42) oligomer was prepared by using chemical synthetic Aβ_(1-42) polypeptide in vitro. The prepared peptide was identified by the assay of Western blotting. In order to confirm the neurotoxicity of the peptide,primary cultured astrocytes from the cortex of rats were treated with different concentrations of Aβ_(1-42) oligomer and the survival rates of the cells were detected via Cell Counting Kit-8( CCK-8) assay. Results: The results indicated that chemical synthetic Aβ_(1-42) oligomer weighed from 4 to188 kd,in which the points with 64 kd were the most. In addition,a few points with 4 kd,a monomer,was also found. Furthermore,the neurotoxicity of Aβ_(1-42) oligomer on astroytes was significantly detected with a manner of dose-dependence. Conclusions: An improved method for preparation and identification of Aβ_(1-42) oligomer has been successfully established,which is important in the research work of Alzheimer's disease.

关键词(KeyWords)： 淀粉样蛋白;阿尔茨海默病;寡聚体;星形胶质细胞;免疫印迹法
amyloid;Alzheimer disease;oligomer;astrocytes;immunoblotting

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81360199);国家自然科学基金(81260173);; 教育部科学技术研究项目(213032A);; 贵州省科技厅国际合作项目[黔科合外G字(2011)7014号];; 贵州省科技计划[黔科合重大专项字(2014)6008号]

作者(Author): 禹文峰,孔欣,官志忠
YU Wenfeng,KONG Xin,GUAN Zhizhong

DOI: 10.19367/j.cnki.1000-2707.2016.08.003

参考文献(References)：

• [1]官志忠.阿尔茨海默病的病理学及生物膜结构与受体改变机制[J].中华病理学杂志,2003(1):69-71.
• [2]Kumar A,Singh A,Ekavali.A review on Alzheimer’s disease pathophysiology and its management:an update[J].Pharmacological Reports,2015(67):195-203.
• [3]刘文娟,戴雪伶,姜招峰.β-淀粉样蛋白神经毒性及其防治策略[J].生命科学,2011(10):1023-1026.
• [4]Anand R,Gill KD,Mahdi AA.Therapeutics of Alzheimer’s disease:past,present and future[J].Neuropharmacology,2014(76):27-50.
• [5]Hardy J.The amyloid hypothesis for Alzheimer’s disease:a critical reappraisal[J].J Neurochem,2009(4):1129-1134.
• [6]Dal Pra I,Chiarini A,Gui L,et al.Do strocytes collaborate with neurons in spreading the‘infectious’Aβand tau rivers of Alzheimer’s disease[J].Neuroscientist,2015(1):9-29.
• [7]Mc Carthy KD,Vellis JD.Preparation of separate astroglial and oligdendroglial cell cultures from rat cerebral tissue[J].Cell Biol,1980(85):890-902.
• [8]Schildge S,Bohrer C,Beck K,et al.Isolation and Culture of Mouse Cortical Astrocytes[J].J Vis Exp,2013(71):1-7.
• [9]汤婷婷,官志忠,禹文峰.SD大鼠大脑皮层星形胶质细胞的体外原代培养[J].贵阳医学院学报,2014(2):158-161.
• [10]张敬军.星形胶质细胞的研究[J].中国药理学通报,2006(7):788-791.
• [11]Klein WL.Aβtoxicity in Alzheimer’s disease:globular oligomers(ADDLs)as new vaccine an drug targets[J].Neurochemistry International,2002(41):345-352.
• [12]官志忠,齐晓岚,欧阳凯,等.阿尔茨海默病发病中β淀粉样蛋白与胆碱能尼古丁受体的相互作用[C].第十三届全国诊断病理暨头颈部及内分泌系统疾病学术研讨会论文集,2012:92-97.
• [13]杨帆,李东风,徐书雯.Aβ1-42寡聚体与纤维体的制备及鉴定[J].中华老年学杂志,2013(33):2076-2078.
• [14]谭现伟.Aβ1-42寡聚体制备方法的研究[D].北京:北京交通大学,2009:1-37.
• [15]Galimberti D,Ghezzi L,Scarpini E.Immunotherapy against amyloid pathology in Alzheimer’s disease[J].J Neurol Sci,2013(1):50-54.
• [16]Selkoe DJ.Alzheimer’s disease is a synaptic failure[J].Science,2002(298):789-791.
• [17]Haass C,Selkoe DJ.Soluble protein oligomer in neurodegeneration:lessons from the Alzheimer's amyloid betapeptide[J].Nat Rev Mol Cell Boi,2007(2):101-112.
• [18]Shankar GM,Li S,Mehta TH,et al.Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory[J].Nat Med,2008(8):837-842.
• [19]Butterfield DA.Amyloidβ-peptide(1-42)-induced oxidative stress and neurotoxicity:implications for neurodegeneration in Alzheimer's disease brain[J].Free Radical Research,2002(12):1307-1313.
• [20]Heneka MT,O'Banion MK.Inflammatory processes in Alzheimer's Disease[J].J Neuroimmunol,2007(1-2):69-91.
• [21]王琦,周海霞.阿尔茨海默病中β淀粉样蛋白对星形胶质细胞的作用[J].中国老年学杂志,2011(18):3643-3645.

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