HER2靶向重组截短型Bid片段融合蛋白对骨肉瘤细胞的促凋亡作用  

The pro-apoptotic effect of HER2 targeted recombinant truncated Bid fusion protein on osteosarcoma cells

作　　者：纪振钢[1] 裘秀春[1] 杨彤涛[1] 龙华[1] 马保安[1] 周勇[1] 张明华[1] 许彦鸣[2] 杨安钢[3] 范清宇[1] 

机构地区：[1]第四军医大学唐都医院骨科中心,陕西西安710038 [2]第四军区大学生物化学与分子生物学教研室,陕西西安710032 [3]第四军区大学免疫学教研室,陕西西安710032

出　　处：《医学研究生学报》2007年第2期120-122,127,I0001,共5页Journal of Medical Postgraduates

基　　金：国家自然科学基金资助项目(批准号:30330610);国家自然科学基金资助项目(批准号:30471988);中国博士后基金资助项目(批准号:2005038259)

摘　　要：目的:观察人上皮细胞生长因子受体2(HER2)靶向重组的活性截短型Bid(tBid)融合蛋白对骨肉瘤SOSP-9607细胞的促凋亡作用。方法:将抗HER2单链抗体基因e23sFv、与绿脓杆菌外毒素(PE)的转膜结构域基因(PEII)和tbid基因连接,构建成immuno-tbid(e23sFv-PEII-tbid)基因,将其克隆入真核表达载体pCMV中,转染SOSP-9607细胞,间接免疫荧光法检测目的蛋白表达和细胞形态学变化,通过AnnexinⅤ染色、流式细胞仪检测观察其促凋亡作用。结果:转染SOSP-9607细胞后,间接免疫荧光染色检测出tB id的表达,SOSP-9607细胞出现明显的固缩,核浓缩等凋亡特征。AnnexinⅤ染色、流式细胞仪检测可见明显的凋亡细胞,凋亡率为42%,对照组细胞仅6%,表明细胞膜表面有磷脂酰丝氨酸外翻,提示重组immuno-tbid基因表达后有促凋亡作用。结论:重组immuno-tbid基因可在转染的SOSP-9607细胞中表达,并诱导其细胞凋亡。Objective：To investigate the pro-apoptotic effect of HER2 targeted recombinant trucated BH3 interacting death agonist（tBid） fusion protein on osteosarcoma SOSP-9607 cells. Methods：Immuno-tbid gene was generated by sequentially fusing the coding sequences of a signal peptide, a singlechain HER2 antibody （e23sFv）, a PE translocation domain （PE aa253-364） and tBid, the immunotbid genes was cloned into a pCMV plasmid and was transfected into SOSP-9607 cells. Expression of recombinant immuno-tbid genes and morphologic changes of SOSP-9607 cells were detected by immunofluorescent staining. Annexin V-FITC staining was measured by flow cytometry, and apoptosis effect induced by overexpression of recombinant tBid genes was analyzed. Results ： The tBid protein was expressed in SOSP-9607 cells were transfected with immuno-tbid genes, followed by cell shrinkage and nuclear condensation. Annexin V-FITC staining revealed that the percentages of apoptotic cells in the transfectants of immuno-tbid genes were 42% , compared to 6% in the control cells, suggesting the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Conclusion ： HER2 targeted recombinant tBid fusion protein can induce apoptosis of SOSP-9607 cells.

关 键 词：截短型Bid片段 人上皮细胞生长因子受体2 融合蛋白 骨肉瘤SOSP-9607细胞 细胞凋亡 

分 类 号：R738.7[医药卫生—肿瘤]