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.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
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)~~
