细胞浆中PrP蛋白的表达及其细胞毒性作用的研究  

Expression of Cytosolic PrP and Analysis of Its Cytotoxic Activities

作　　者：王新[1,2] 董辰方[2] 石琦[2] 石崧[2] 王桂荣[2] 雷艳君[2,3] 安润[2,3] 徐琨[2,3] 姜慧英[2] 韩俊[2] 赵玉军[1] 董小平[2] 

机构地区：[1]沈阳农业大学畜牧兽医学院,沈阳110161 [2]中国疾病预防控制中心病毒病预防控制所,传染病预防控制国家重点实验室,北京100052 [3]西安交通大学医学院,西安710061

出　　处：《病毒学报》2008年第4期277-281,共5页Chinese Journal of Virology

基　　金：国家科技攻关项目(2006BAD06A13-2);国家973项目(2007CB310505);国家自然科学基金委项目(No.30500018、30571672)资助

摘　　要：为了探讨在胞浆中表达的PrP的理化特征以及对细胞活性的影响,我们构建了胞浆型PrP(CytoPrP)真核表达质粒,瞬时转染人神经母细胞瘤SH-SY5Y细胞,通过蛋白酶敏感性实验检测CytoPrP及其蛋白酶抗性,利用MTT和Trypan Blue细胞计数检测CytoPrP的细胞毒性作用。结果显示,CytoPrP在胞浆内的存在受蛋白酶抑制剂的控制;与野生型PrP相比,CytoPrP具有相对较强的蛋白酶K(PK)抗性。MTT和细胞计数实验均显示,Cyto-PrP的存在可诱导明显的细胞毒性效应,而CytoPrP的细胞毒性作用受蛋白酶抑制剂含量的影响,呈剂量依赖关系。上述结果为研究CytoPrP在朊病毒病的发病机制中的意义提供了一定的科学数据。In order to study the physicochemical characteristics of cytosolic PrP （CytoPrP） and evaluate its possible influence on cell viability, a recombinant plasmid expressing human CytoPrP eukaryoticly was constructed and transfected into human neuroblastoma cell line SH-SY5Y transiently. Proteinase-resistant activities of CytoPrP were evaluated by a proteinase K （PK） digestion and cytotoxic effects of CytoPrP were tested by MTT assay and Trypan Blue cell-counting. The presence of CytoPrP in cytoplasm after transfection was controlled by the presence of protease inhibitor. Compared with wild-type PrP, CytoPrP possessed relatively stronger PK-resistant activities. Obvious cytotoxic effects were observed in the cells after inducement of CytoPrP in cytoplasm by protease inhibitor, showing a dose-dependent manner. The results provide useful scientific evidences for further studies of potential role of CytoPrP in pathological mechanism of prion disease.

关 键 词：朊病毒 胞浆PrP 细胞毒作用 蛋白酶抗性 

分 类 号：R373.9[医药卫生—病原生物学]