持续释放人血管抑素的基因修饰化工程细胞h E/293细胞株的建立  

Construction of gene engineered hE/293 cells continuously releasing human endostatin

作　　者：赵卉[1] 潘静坤[1] 罗芸[1] 田磊[1] 薛毅珑[1] 

机构地区：[1]中国人民解放军总医院老研所细胞室,北京市100853

出　　处：《世界华人消化杂志》2007年第12期1370-1375,共6页World Chinese Journal of Digestology

摘　　要：目的:在人胚肾HEK293细胞中转染真核表达载体pSNA2/hEndostatin(hEndostatin,人血管抑素),建立能稳定分泌hES的基因工程细胞株.方法:将含有IL-2分泌肽的人endostatin(ES)全长cDNA插入真核表达载体pSNA2,产生重组质粒pSNA2/hEndostatin;利用阳离子脂质体介导将其转染入HEK293细胞中;用G418筛选出阳性克隆细胞,将其命名为hE/293细胞.用Western blot法检测hE/293细胞培养上清中分泌的hES蛋白.血管内皮细胞(ECV304)增殖抑制试验及鸡胚尿囊膜试验观察其分泌的hES蛋白的抗增殖活性.结果:经过双酶切和DNA测序证实构建出了含hES基因的真核表达载体.通过G418抗性筛选筛选出稳定表达hES的细胞株3株,将其命名为hE/293细胞;Western blot检测该细胞株培养上清中存在分子量为20 kDa的ES蛋白;ECV304增殖抑制试验显示,与HEK293细胞组相比,hE/293细胞组分泌的ES蛋白对bFGF刺激的血管内皮细胞增殖有明显的抑制作用(48h:0.125±0.007 vs 0.159±0.020,P<0.01;72 h:0.088±0.016 vs 0.249±0.070,P<0.01);鸡胚尿囊膜试验证实hE/293细胞分泌的ES蛋白可以抑制鸡胚尿囊膜血管生长.结论:所构建的hE/293细胞株可以稳定的分泌hES蛋白,并能抑制ECV304细胞生长及鸡胚尿囊膜血管生长.AIM： To express the recombinant eukaryotic expression vector containing human endostatin gene in human embryonic kidney HEK293 cells, and construct a cell line continuously secreting human endostatin （hES）. METHODS： Human endostatin cDNA containing interleukin-2 （IL-2） secreting peptide was cloned into eukaryotic expression plasmid pSNA2 to construct the recombinant plasmid pSNA2/endostatin. The plasmid pSNA2/endostatin was transfected into HEK293 cells by cationic liposome. The positive cell clones were selected by G418 and then named hE/293 cells. The expression of endostatin protein was analyzed by Western blot. The release of biologically active endostatin was confirmed using assays of ECV304 proliferation and the angiogenesis experiment of chicken chorioallantoic membrane （CAM）. RESULTS： Enzyme digestion and sequence analysis confirmed that the eukaryotic expression vector pSNA2/endostatin had been suc- cessfully constructed. After G418 selection, 3 strains of cells stably expressing hES were obtained, named as hE/293 cells. Western blot showed that ES protein with a molecular weight of 20 kDa existed in the supernatant of hE/293 cells. No endostatin expression was found in the control cells. The assays of ECV304 proliferation showed that the supernatant of hE/293 cells inhibited ECV304 cell proliferation induced by basic fibroblast growth factor in comparison with that of HEK29 cells （48 h： 0.125 ± 0.007 vs 0.159 ± 0.020, P 〈 0.01; 72 h： 0.088 ± 0.016 vs 0.249 ± 0.070, P 〈 0.01）. There were fewer blood vessels in the CAM treated with ES protein. CONCLUSION： Target cell line hE/293 is successfully constructed which it can stably secret hES protein, inhibit the ECV304 cell proliferation and CAM angiogenesis.

关 键 词：人内皮抑素 真核表达载体 人胚胎肾细胞 hES细胞株 WESTERN BLOT 

分 类 号：R73-3[医药卫生—肿瘤]