中华神经科杂志
中華神經科雜誌
중화신경과잡지
Chinese Journal of Neurology
2010年
9期
632-636
,共5页
樊彩妮%丁健青%陈生弟%汤荟冬
樊綵妮%丁健青%陳生弟%湯薈鼕
번채니%정건청%진생제%탕회동
淀粉样β蛋白前体%阿尔茨海默病%毒理学%细胞系,肿瘤
澱粉樣β蛋白前體%阿爾茨海默病%毒理學%細胞繫,腫瘤
정분양β단백전체%아이자해묵병%독이학%세포계,종류
Amyloid beta-protein precursor%Alzheimer disease%Toxicology%Cell line,tumor
目的 体外探讨β淀粉样前体蛋白(amyloid precursor protein,APP)羧基端肽(APPC31)对小鼠神经母细胞瘤(Neuro2a)细胞的毒性作用及在Aβ42致毒性中的作用.方法 分别将空载体质粒和过表达APPC31质粒通过脂质体转染法瞬时转染Neuro2a细胞,48 h后四甲基偶氮唑盐比色(MTT)法检测细胞活力,同时利用4',6-联脒-2-苯基吲哚(DAPI)核染色观察细胞形态,与空载体转染细胞组对照,观察APPC31对细胞有无毒性作用;然后通过相同方法,在Neuro2a细胞中,分别将过表达的空载体质粒、野生型APP695质粒、APP(D664A)质粒、APP氨基端长肽(APP△C31)质粒和APPC31质粒瞬时转染Neuro2a细胞,24 h后加Aβ42(10 μmol/L)共孵育24 h,MTT法检测细胞活力及DAPI核染色观察细胞形态,与空载体转染细胞组对照,观察Aβ42处理条件下APPC31的毒性作用;质粒转染细胞后通过免疫荧光检测目的 基因表达情况.结果 在无Aβ42共孵育时,空载体质粒转染组和APPC31质粒转染组细胞活力分别为:0.81±0.10、0.88±0.12,两组之间比较差异无统计学意义(t=-0.78,P=0.48),细胞核形态亦无明显凋亡或死亡改变;Aβ42(10 μmol/L)共孵育条件下,无转染组、空载体质粒组、野生型APP695质粒组、APP(D664A)质粒组和APPC31质粒组细胞活力分别为:0.82±0.01、0.78±0.03、0.55±0.04、0.81±0.04、0.78±0.02、0.54±0.02,且与空载体质粒组相比,野生型APP695质粒组及APPC31质粒组细胞活力明显下降(F=47.53,P＜0.05),细胞核呈明显浓缩、染色加深改变.结论 体外Neuro2a细胞单独过表达一定水平的APPC31并不能致细胞死亡;但此短肽可增强Aβ42的细胞毒性作用,为进一步明确阿尔茨海默病发病机制提供了一定的实验依据.
目的 體外探討β澱粉樣前體蛋白(amyloid precursor protein,APP)羧基耑肽(APPC31)對小鼠神經母細胞瘤(Neuro2a)細胞的毒性作用及在Aβ42緻毒性中的作用.方法 分彆將空載體質粒和過錶達APPC31質粒通過脂質體轉染法瞬時轉染Neuro2a細胞,48 h後四甲基偶氮唑鹽比色(MTT)法檢測細胞活力,同時利用4',6-聯脒-2-苯基吲哚(DAPI)覈染色觀察細胞形態,與空載體轉染細胞組對照,觀察APPC31對細胞有無毒性作用;然後通過相同方法,在Neuro2a細胞中,分彆將過錶達的空載體質粒、野生型APP695質粒、APP(D664A)質粒、APP氨基耑長肽(APP△C31)質粒和APPC31質粒瞬時轉染Neuro2a細胞,24 h後加Aβ42(10 μmol/L)共孵育24 h,MTT法檢測細胞活力及DAPI覈染色觀察細胞形態,與空載體轉染細胞組對照,觀察Aβ42處理條件下APPC31的毒性作用;質粒轉染細胞後通過免疫熒光檢測目的 基因錶達情況.結果 在無Aβ42共孵育時,空載體質粒轉染組和APPC31質粒轉染組細胞活力分彆為:0.81±0.10、0.88±0.12,兩組之間比較差異無統計學意義(t=-0.78,P=0.48),細胞覈形態亦無明顯凋亡或死亡改變;Aβ42(10 μmol/L)共孵育條件下,無轉染組、空載體質粒組、野生型APP695質粒組、APP(D664A)質粒組和APPC31質粒組細胞活力分彆為:0.82±0.01、0.78±0.03、0.55±0.04、0.81±0.04、0.78±0.02、0.54±0.02,且與空載體質粒組相比,野生型APP695質粒組及APPC31質粒組細胞活力明顯下降(F=47.53,P＜0.05),細胞覈呈明顯濃縮、染色加深改變.結論 體外Neuro2a細胞單獨過錶達一定水平的APPC31併不能緻細胞死亡;但此短肽可增彊Aβ42的細胞毒性作用,為進一步明確阿爾茨海默病髮病機製提供瞭一定的實驗依據.
목적 체외탐토β정분양전체단백(amyloid precursor protein,APP)최기단태(APPC31)대소서신경모세포류(Neuro2a)세포적독성작용급재Aβ42치독성중적작용.방법 분별장공재체질립화과표체APPC31질립통과지질체전염법순시전염Neuro2a세포,48 h후사갑기우담서염비색(MTT)법검측세포활력,동시이용4',6-련미-2-분기신타(DAPI)핵염색관찰세포형태,여공재체전염세포조대조,관찰APPC31대세포유무독성작용;연후통과상동방법,재Neuro2a세포중,분별장과표체적공재체질립、야생형APP695질립、APP(D664A)질립、APP안기단장태(APP△C31)질립화APPC31질립순시전염Neuro2a세포,24 h후가Aβ42(10 μmol/L)공부육24 h,MTT법검측세포활력급DAPI핵염색관찰세포형태,여공재체전염세포조대조,관찰Aβ42처리조건하APPC31적독성작용;질립전염세포후통과면역형광검측목적 기인표체정황.결과 재무Aβ42공부육시,공재체질립전염조화APPC31질립전염조세포활력분별위:0.81±0.10、0.88±0.12,량조지간비교차이무통계학의의(t=-0.78,P=0.48),세포핵형태역무명현조망혹사망개변;Aβ42(10 μmol/L)공부육조건하,무전염조、공재체질립조、야생형APP695질립조、APP(D664A)질립조화APPC31질립조세포활력분별위:0.82±0.01、0.78±0.03、0.55±0.04、0.81±0.04、0.78±0.02、0.54±0.02,차여공재체질립조상비,야생형APP695질립조급APPC31질립조세포활력명현하강(F=47.53,P＜0.05),세포핵정명현농축、염색가심개변.결론 체외Neuro2a세포단독과표체일정수평적APPC31병불능치세포사망;단차단태가증강Aβ42적세포독성작용,위진일보명학아이자해묵병발병궤제제공료일정적실험의거.
Objective To investigate the toxic effect of the carboxyl-terminal peptide of β-amyloid precursor protein (APPC31) on Neuro2a cells as well as its role in the toxic process in Neuro2a cells induced by Aβ42 in vitro.Methods The plasmid vector and the APPC31 construct were transiently transfected into Neuro2a cells respectively by lipofectamine 2000.The viability of the cells was measured by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 48 h after transfection,and their morphocytology was observed by 4', 6-diamidino-2-phenylindole (DAPI) nucleus staining.Afterword different constructs including vector, WTAPP695, APP( D664A), the amino-terminal peptide of β-amyloid precursor protein (APP△C31) and APPC31 were transiently transfected into Neuro2a cells respectively via the same method.At 24 h after transfection Aβ42 was added into the culture medium of Neuro2a cells with the desired concentration for another 24 h for cell studies.The viabihty and morphocytology of the cells were measured by using the MTT assay and DAPI nucleus staining, respectively.Results When incubated in the absence of Aβ42, the viability of cells transfected with vector and APPC31construct were 0.81 ±0.10 and 0.88 ±0.12 respectively, and accordingly there was no significant difference between the these two groups (t = - 0.78, P = 0.48 ); meanwhile no obvious cell nuclear morphological changes of apoptosis or death occurred.However when incubated in the presence of Aβ42, the viability of cells transfected with vector, WTAPP695, APP( D664A), APP△C31 and APPC31 constructs were 0.82 ±0.01, 0.78 ±0.03, 0.55 ±0.04, 0.81 ±0.04, 0.78 ±0.02 and 0.54 ±0.02 respectively.The viability of cells transfected with WTAPP construct and APPC31 construct decreased significantly ( F = 47.53, P ＜0.05) compared with the control group, meanwhile cells displayed condensed nuclear and even nuclear fragmentation.Conclusions In vitro, over-expression of a certain level of APPC31 in Neuro2a cells fails in causing cell death, but this short peptide enhances cytotoxicity induced by Aβ42 in Neuro2a cells.Thus,these results provide the experimental basis for the further explication of the pathogenesis of Alzheimer's disease.
