摘要
朊病毒是一类能引起人或动物神经退行性疾病的感染因子。蛋白质错误折叠循环扩增(Protein misfolding cy-clic amplification,PMCA)是一种常见的朊病毒体外扩增技术。白藜芦醇是一种具有较高研究价值的植物抗菌素。为了建立SMB-S15细胞中朊病毒的PMCA体系,并探索PMCA技术在评价白藜芦醇抗朊病毒作用的意义,本研究以朊病毒感染细胞系SMB-S15细胞匀浆为种子,健康小鼠脑组织匀浆为底物,建立PMCA技术平台。将不同浓度的白藜芦醇导入PMCA体系,通过Western Blot方法检测PMCA产物中异常朊蛋白(PrPSc)的生成和变化。结果显示:SMB-S15细胞中PrPSc可以在建立的PMCA体系有效地复制。新生成的PrPSc分子的糖基化特征与原始细胞中的PrPSc不同,其双糖基化分子比例明显增加。同时也有别于羊瘙痒因子139A和ME7感染小鼠脑组织中的PrPSc分子。加入白藜芦醇后,PrPSc在PMCA体系中的复制明显被抑制,呈现出剂量依赖效应,其EC50约为4.616μM。这些结果表明:SMB-S15细胞中的PrPSc可以通过PMCA大量扩增,建立的PMCA技术可有效地反映白藜芦醇对PrPSc在体外复制的抑制作用。
Prions are infectious agents that can cause neurodegenerative diseases in humans and animals. Protein misfolding cyclic amplification(PMCA)is a common prion amplification technique in vitro. Resveratrol is a plant antibiotic with high research value. The purpose of this study was to establish PMCA methodology for the replication of PrPScin SMB-S15 cells and to evaluate the anti-prion activity of resveratrol by PMCA. PMCA methodology was established using the lysates of SMB-S15 cells as seeds and brain homogenates of healthy C57 mice as substrates. Various amounts of resveratrol were introduced into the PMCA methodology,and production and changes of PrPScamounts in PMCA products were analyzed by PrP-specific Western Blot after digestion with proteinase K(PK). The PrPScderived from SMB-S15 cells replicated in the established PMCA method. Glycosylation patterns of the newly formed PrPScin PMCA products were different from the original one in SMB-S15 cells(showing increase of di-glycosylated molecules)and they were also different compared with the patterns of PrPScin the brain tissues collected from mice infected with the scrapie agents 139 A and ME7. Addition of resveratrol into PMCA reactions inhibited PrPScpropagation efficiently in a dose-dependent manner. The EC50 of resveratrol for inhibiting PrPScreplication in PMCA was 4.616 μM. These results suggest that PrPSc from SMB-S15 cells can be amplified in PMCA. The established PMCA methodology can reflect the inhibitory effect of resveratrol on PrPScamplification in vitro.
引文
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