阿尔茨海默病中β淀粉样前体蛋白羧基端短肽C31与自噬的相关性研究  

Correlation study of β-amyloid precursor protein carboxy-terminal peptide C31 in Alzheimer disease and autophagy

作　　者：刘敏[1] 马建芳[1] 刘晓黎[1] 汤荟冬[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院神经内科神经病学研究所,200025

出　　处：《神经疾病与精神卫生》2015年第1期15-21,F0003,共8页Journal of Neuroscience and Mental Health

基　　金：国家自然科学青年基金（81200979）

摘　　要：目的 探讨β淀粉样前体蛋白羧基端短肽C31的毒性作用和自噬的关系,以及自噬是否介导C31的清除.方法 首先构建p3 xFLAG—CMV—10—mCherry-C31（C31）质粒和p3xFLAG-CMV-10-mCherry（Vector）质粒（对照）,采用脂质体转染法转染入SH-SY5Y和APPsw稳转的HEK293（APPsw HEK293）细胞系中,并通过Western blot方法验证其表达;采用MTT法检测C31和Vector质粒瞬时转染48 h后的细胞存活率;分别予以不同的自噬调节药物,Western blot检测自噬相关蛋白（LC3）和C31水平.最后,通过免疫共沉淀法检测C31是否可以和LC3结合,并采用免疫荧光检测C31是否可以与LC3共定位,以进一步验证自噬是否为介导C31降解的关键途径.结果 在SH-SY5Y和APPsw HEK293细胞系中,C31质粒转染组细胞活力显著低于对照组（P＜0.05）;转染C31组与对照组自噬水平无明显差异;促自噬情况下（雷帕霉素和饥饿处理）,LC3—Ⅱ的水平比未加药处理组略增高,但未达到统计学意义,C31水平相对于空载水平差异无统计学意义;自噬抑制剂3-甲基腺嘌呤（3-MA）作用于APPsw HEK293细胞后,LC3—Ⅱ水平有轻度降低,但未达到统计学意义,C31水平改变也未达到统计学意义;加入自噬体和溶酶体融合抑制剂氯喹（CQ）和氯化铵（NH4Cl）后,细胞自噬水平有明显的增高,且CQ的作用最为明显,C31水平相对于空载水平明显增高（P＜0.05）.在SH—SY5Y细胞中,结果类似,但CQ和NH4Cl作用后,C31水平相对于空载水平无统计学差异.免疫荧光显示C31通过与自噬相关蛋白LC3结合,从而锚定到自噬体上,而参与到自噬代谢过程.结论 C31在SH-SY5Y细胞和APPsw HEK293细胞中均能产生细胞毒性作用.过表达C31不改变细胞的自噬水平.自噬途径介导C31的清除,表明自噬可能在阿尔茨海默病中起到保护作用.Objective To investigate the relationship between the toxic effect of β--amyloid precursor protein carboxy-terminal peptide C31 and autophagy, Specifically, to explore the role of autoph- agy in the degradation of C31 and the relevant mechanism. Methods First constructed p3xFLAG-CMV - 10 - mCherry （control） and p3 xFLAG- CMV- 10 - mCherry- C31 （ C31 ）, using lipofectioon transfected the two constructs into SH--SY5Y and APPsw HEN293 cells respectively. Then verified its expression by Western blot. MTT method was used to detect C31 and the cell survival rate through vector plasmids were transiently transfected for 48h. Given different autophagy regulating drugs we used western blot to detect the level of autophagy--related protein （LC3） and C31. Finally, whether C31 and LC3 could combined by immune coprecipitation, and using immunofluorescence to detect whether C31 could co--locate with LC3, to further verify whether autophagy is the critical path of C31- mediated degradation. Results In the SH--SY5Y and APPsw HEK293 cell lines, the viability of cells transfected with the C31 plasmid was significantly lower than vector group （P 〈 0.05）. There was no significant difference in the level of autophagy between the C31 and control group in both cell lines; Under the conditions of promoting autophagy （rapamycin and starvation）, the level of LC3-II was slightly increased, but didn＇t reach the statistical significance and the C31 levels were not statistically changed. When using autophagy inhibitors, 3-methyl adenine （3-MA） acting on the APPsw HEK293 ceils, both LC3-II and C31 levels were not statistically changed, but LC3-II levels were mildly reduced. After adding autophagy and lysosome fusion inhibitors chloroquine（CQ） and ammonium chloride（NH4C1）, autophagy levels significantly increased, and the role of CQ is the most obvious, C31 levels were statistically increased （P 〈 0.05）. In SH--SY5Y cells, the results were similar, but after adding the CQ and NH4C1, the C31 levels were

关 键 词：阿尔茨海默病 自噬 C31 LC3 

分 类 号：R749.16[医药卫生—神经病学与精神病学]