过表达α-synuclein的细胞毒性及对损伤性刺激的影响  

Cytotoxicity of α-synuclein over-expression and the effects on injury stimulus

作　　者：金金[1] 苑玉和[1] 杨波[1] 孙建栋[1] 陈乃宏[1] 

机构地区：[1]中国医学科学院北京协和医学院药物研究所,北京100050

出　　处：《中国老年学杂志》2010年第6期760-763,共4页Chinese Journal of Gerontology

基　　金：国家自然科学基金(30973887);国家重大新药创制〔科技重大专项〕(2008ZX09101);教育部博士点基金(20070023037);国家高技术研究发展计划〔863计划重点项目〕(2006AA020501);国家自然科学基金广东自然科学联合基金(U0832008)

摘　　要：目的构建过表达α-synuclein的细胞模型,研究过表达α-synuclein的细胞毒性作用及其分子机制,为揭示帕金森病(PD)可能的发病机制和寻找治疗药物潜在靶点提供依据。方法脂质体法转染及过表达α-synuclein稳定株的构建,MTT法、PI染色、荧光显微镜观察用于检测过表达α-synuclein对细胞活性和分化、撤血清及MPP+损伤的影响,荧光素酶报告基因检测和Western印迹检测用于研究过表达α-synuclein对NF-κB信号通路及相关蛋白的影响。结果过表达α-synuclein对细胞的分化表型无明显影响,但能明显降低DA能细胞存活率,并显著增加撤血清损伤及MPP+毒性对细胞的损伤程度。过表达α-synuclein能够明显加重MPP+对NF-κB转录激活活性的抑制作用,其机制可能与抑制IκB-α的磷酸化有关。结论基于遗传背景与环境因素相结合的PD研究模型能够更好地拟合其病理过程,更加科学合理。NF-κB信号通路活性的抑制可能是PD发病的又一新机制,针对该通路上相关蛋白的深入研究可能成为PD治疗药物开发的新型研究方向。Objective To construct α-synuclein over-expressed cell model and study cytotoxicity and mechanisms of α-synuclein overexpression,which might reveal possible pathogenesis of Parkinson＇s disease （PD） and provide novel targets for drug research and development （RD） of PD therapy.Methods The recombinant pEGFP-N1-α-synuclein vectors were transfected via LipofectamineTM 2000 to generate α-synuclein over-expressed cells that could be observed under a fluorescence microscope.Then,the transgenic cells were subjected to Western blot assay,MTT assay,PI staining and luciferase assay respectively.Results Over-expression of α-synuclein did not influence the differentiation of PC12 cells,however,could decrease cell viability significantly and aggravate the injury of serum starvation or neurotoxin MPP＋.Furthermore,α-synuclein over-expression could aggravate MPP＋ induced inhibition of transcription factor NF-κB mediated luciferase expression by inhibition of IκB-α phosphorylation.Conclusions Combinations of genetic factor and environmental factor in experimental models make the study of PD pathogenesis more scientific and rational.Moreover,the inhibition of NF-κB signaling pathway is regarded as a potential mechanism of PD pathogensis.Thus,a further study of proteins changes presented in the signal pathway should be necessary for novel targets discovery of PD therapy and the followed drug RD.

关 键 词：帕金森病 Α-SYNUCLEIN 细胞毒性 MPP+ NF-ΚB 

分 类 号：R965.1[医药卫生—药理学]