摘要
目的观察hSav1蛋白高表达对Mst1介导的细胞凋亡的影响，探讨hSav1在Mst1介导的细胞凋亡中的作用。方法构建蛋白hSav1的载体pCMV—HA—hSav1与蛋白Mst1的载体pcDNA／4TO—Flag-Mst1，共转染HeLa细胞。采用相应抗体做二者的免疫共沉淀，以证实蛋白hSav1和Mst1之间的相互作用；应用细胞免疫荧光共定位，进一步证实二者之间的相互作用。在HeLa细胞中，分别单独或同时转染质粒pcDNA／4TO-Flag—Mst1和pCMV—HA—hSav1，转染36h后，加入凋亡诱导剂顺铂（50μmol／L）作用14h。应用磷脂结合蛋白碘化丙啶（Annexin V／PI）法检测细胞凋亡，观察蛋白hSav1高表达对Mst1介导的细胞凋亡的影响。结果载体pCMV-HA—hSav1与pcDNA／4TO—Flag-Mst1构建成功，测序结果表明无突变或缺失。免疫共沉淀结果显示，蛋白hSav1可以在Mst1抗体的免疫沉淀物中检测出来，蛋白Mst1可以在hSav1抗体的免疫沉淀物中检测出来。细胞免疫荧光的结果显示，二者的荧光在细胞内存在共定位，并可充分融合。HeLa细胞中，单独Mst1蛋白高表达组的细胞凋亡率为24．5％±2．4％，单独hSav1蛋白高表达组与对照组比较，无明显细胞凋亡。蛋白Mst1和hSav1高表达组的细胞凋亡率为39．3％±4．0％，差异有统计学意义（P〈0．05）。结论蛋白hSav1与Mst1在HeLa细胞内存在相互作用，蛋白hSav1高表达可明显促进由蛋白Mst1介导的细胞凋亡。
Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSavl and pcDNA/4TO-Flag-Mstl were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSavl, Mstl and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSavl and/or pcDNA/4TO-Flag-Mstl were transfeeted into HeLa cells, and 36 hours later cisplatin （50 μmoL/L） as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSavl and pcDNA/4TO-Flag-Mstl were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, coxpression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone （ 24.5% ± 2.4% vs. 39.3% -4.0%, P 〈 0.05 ）. Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mstl and augments Mstl-mediated apoptosis.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2009年第7期481-484,共4页
Chinese Journal of Oncology
基金
教育部新世纪优秀人才支持计划（NCET-04-0699）
国家自然科学基金（30872472
30800569）
关键词
hSav1
Mst1
蛋白相互作用
细胞凋亡
HELA细胞
Human salvador 1
Mammalian sterile 20-like kinase 1
Protein interactions
Apoptosis
HeLa cells
