西安交通大学学报（医学版）
西安交通大學學報（醫學版）
서안교통대학학보（의학판）
JOURNAL OF XI'AN JIAOTONG UNIVERSITY(MEDICAL SCIENCES)
2010年
1期
32-35,46
,共5页
徐琨%王新%田婵%石崧%王桂荣%石琦%周瑞敏%姜惠英%楚雍烈%董小平
徐琨%王新%田嬋%石崧%王桂榮%石琦%週瑞敏%薑惠英%楚雍烈%董小平
서곤%왕신%전선%석숭%왕계영%석기%주서민%강혜영%초옹렬%동소평
doppel蛋白%朊蛋白%细胞毒性作用%细胞凋亡
doppel蛋白%朊蛋白%細胞毒性作用%細胞凋亡
doppel단백%원단백%세포독성작용%세포조망
doppel protein%PrP%cytotoxicity%apoptosis
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24h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Annexin V/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.
目的 體外觀察細胞瞬時錶達的人Dpl(doppel)蛋白對人神經母細胞瘤SH-SY5Y細胞的作用,併與其結構類似蛋白--截短型PrP(PrPΔ32-121)加以比較.方法 通過PCR方法穫得人PRND基因及截短型PRNP基因併剋隆至真覈錶達載體,瞬時轉染SH-SY5Y細胞後,觀察蛋白錶達及其定位情況;採用MTT實驗檢測轉染細胞的生長狀態;用流式細胞儀、Western blot等方法檢測轉染細胞的凋亡狀態.結果 兩種蛋白均可在轉染細胞中錶達,併存在于細胞膜上;MTT結果顯示,在轉染24h後均齣現明顯的細胞毒性作用;細胞凋亡相關實驗髮現,轉染細胞Annexin V/PI雙染暘性細胞數量增多,pro-casepase-3和Bcl-2因子水平降低.結論 瞬時錶達的Dpl蛋白與截短型PrP可產生類似的神經細胞毒性作用,併誘導啟動細胞凋亡過程.
목적 체외관찰세포순시표체적인Dpl(doppel)단백대인신경모세포류SH-SY5Y세포적작용,병여기결구유사단백--절단형PrP(PrPΔ32-121)가이비교.방법 통과PCR방법획득인PRND기인급절단형PRNP기인병극륭지진핵표체재체,순시전염SH-SY5Y세포후,관찰단백표체급기정위정황;채용MTT실험검측전염세포적생장상태;용류식세포의、Western blot등방법검측전염세포적조망상태.결과 량충단백균가재전염세포중표체,병존재우세포막상;MTT결과현시,재전염24h후균출현명현적세포독성작용;세포조망상관실험발현,전염세포Annexin V/PI쌍염양성세포수량증다,pro-casepase-3화Bcl-2인자수평강저.결론 순시표체적Dpl단백여절단형PrP가산생유사적신경세포독성작용,병유도계동세포조망과정.
Objective To observe the biological activities of human doppel (Dpl) protein transiently expressed and Dpl-like protein PrPΔ32-121 on a human neuroblastoma cell line SH-SY5Y. Methods Recombinant mammalian expression plasmids containing human PRND gene and truncated PrPΔ32-121 fragment were generated by PCR. The expression and location of Dpl and PrPΔ32-121 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 PrPΔ32-121 protein were expressed and mainly located on the cell membrane. Remarkable cytotoxicity was detected on SH-SY5Y cells after 24 h transfection. Meanwhile, more Annexin V/PI positively-stained cells as well as lower levels of cellular pro-caspase-3 and Bel-2 were detected in the cells receiving Dpl and PrPΔ32-121 expressing plasmids. Conclusion Dpl protein transiently expressed and PrPΔ32-121 can lead to the similar neural cytotoxicity, probably triggering the cell apoptosis program.
