人doppel蛋白及其类似蛋白PrPΔ32-121对SH-SY5Y细胞的细胞毒性作用  

Cytotoxicity of human doppel protein and Dpl-like protein PrPΔ32-121 to SH-SY5Y cells

作　　者：徐琨[1,2] 王新[2] 田婵[2] 石崧[2] 王桂荣[2] 石琦[2] 周瑞敏[2] 姜惠英[2] 楚雍烈[1] 董小平[2] 

机构地区：[1]西安交通大学医学院病原生物学与免疫学系,陕西西安710061 [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

出　　处：《西安交通大学学报（医学版）》2010年第1期32-35,46,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家科技攻关项目(No.2006BAD06A13-2);国家973项目(No.2007CB310505);国家自然科学基金委项目(No.30800975);科研院所基金(No.2008EG150300)~~

摘　　要：目的体外观察细胞瞬时表达的人Dpl(doppel)蛋白对人神经母细胞瘤SH-SY5Y细胞的作用,并与其结构类似蛋白——截短型PrP(PrPΔ32-121)加以比较。方法通过PCR方法获得人PRND基因及截短型PRNP基因并克隆至真核表达载体,瞬时转染SH-SY5Y细胞后,观察蛋白表达及其定位情况;采用MTT实验检测转染细胞的生长状态;用流式细胞仪、Western blot等方法检测转染细胞的凋亡状态。结果两种蛋白均可在转染细胞中表达,并存在于细胞膜上;MTT结果显示,在转染24 h后均出现明显的细胞毒性作用;细胞凋亡相关实验发现,转染细胞AnnexinV/PI双染阳性细胞数量增多,pro-casepase-3和Bcl-2因子水平降低。结论瞬时表达的Dpl蛋白与截短型PrP可产生类似的神经细胞毒性作用,并诱导启动细胞凋亡过程。Objective To observe the biological activities of human doppel （Dpl） protein transiently expressed and Dpl-like protein PrP△32-121 on a human neuroblastoma cell line SH-SY5Y. Methods Recombinant mammalian expression plasmids containing human PRND gene and truncated PrP△32-121 fragment were generated by PCR. The expression and location of Dpl and PrP△32-121 post-transfection were observed by IFA. The cytotoxicity was measured by MTT analysis. Cellular apoptosis was investigated by flow cytometry and Western blot. Results Both Dpl and PrP△32-121 protein were expressed and mainly located on the cell membrane. Remarkable cytotoxicity was detected on SH-SY5Y cclls after 24 h transfection. Meanwhile, more Annexin V/PI positively stained cclls as wcll as lower levels of cellular pro-caspase-3 and Bel-2 were detected in the cells receiving Dpl and PrP△32-121 expressing plasmids.Conclusion Dpl protein transiently expressed and PrP△32-121 can lead to the similar neural cytotoxicity, probably triggering thc cell apoptosis program.

关 键 词：doppel蛋白 朊蛋白 细胞毒性作用 细胞凋亡 

分 类 号：R373[医药卫生—病原生物学]