摘要
目的:构建α-突触核蛋白(α-synuclein,SNCA)和SNCA突变基因(SNCAmu)过表达转染293T细胞,观察内质网应激及细胞凋亡。方法:应用PCR技术扩增目的基因并插入慢病毒载体质粒,重组质粒的构建及包装293T细胞,转染24 h后用荧光显微镜观察绿色荧光蛋白(green fluorescent protein,GFP)的表达。MTT法检测未转染的293T细胞组(CON组)、转染空质粒的293T细胞组(NC组)、转染SNCA基因质粒的293T细胞组(OE组)和转染SNCAmu基因质粒的293T细胞组(OEm组),观察各组细胞病毒转染后不同时间(1、2、3、4、5 d)细胞增殖的情况。Western blotting法测定SNCA和SNCAmu诱导caspase-12的蛋白表达。结果:成功构建了pGC-FU-SNCA-GFP和pGC-FU-SNCAmu-GFP质粒及转染293T细胞,CON组、NC组、OE组和OEm组不同时间的细胞增殖具有统计学意义,SNCA和SNCAmu可能诱导293T细胞caspase-12的蛋白表达。结论:SNCA诱导caspase-12的内质网应激凋亡途径,可能是帕金森病致病机制之一。
Objective:Construction and identification of human SNCA and SNCAmu gene lentiviral vector,transfection into the 293T cells,and induce the endoplasmic reticulum stress.Methods:The target gene was amplified by PCR and inserted into lentiviral vector.The recombinant plasmid was constructed and packaged in 293T cells.The expression of GFP was observed by fluorescence microscope 24 hours after transfection.MTT method was used to detect the proliferation of cells in CON group,NC group,OE group and OEm group at different time points(1、2、3、4、5 days).Western blotting measured the expression of caspase-12 induced by SNCA and SNCAmu.Results:Pgc-FU-SNCA-GFP and pGC-FU-SNCAmu-GFP were successfully constructed and transfected into 293T cells.At different time points(1、2、3、4、5),the proliferation of cells in CON group,NC group,OE group and OEm group has significance in statistics.SNCA and SNCAmu induced the expression of caspase-12 in 293T cells.Conclusion:SNCA induces caspase-12 of endoplasmic reticulum stress apoptosis pathway,which may be one of the pathogenic mechanisms of Parkinson’s disease.
作者
沈原
黄树其
张凯
SHEN Yuan;HUANG Shu-qi;ZHANG Kai(The Department of Neurology,Shanghai Tianyou Hospital,Shanghai,200331,China)
出处
《神经药理学报》
2021年第2期1-4,10,共5页
Acta Neuropharmacologica
基金
上海市卫生健康委员会资助项目(NO.2020LP024)。
关键词
Α-突触核蛋白
帕金森病
慢病毒转染
内质网应激
SNCA
Parkinson’s disease
lentiviral transfection
endoplasmic reticulum stress
