人表皮生长因子受体显性负性突变体对胃癌细胞体外生长能力的影响  

Effect of dominant negative epidermal growth factor receptor on growth of gastric cancer cells in vitro

作　　者：廖刚[1] 王子卫[1] 张能[2] 董浦江[3] 汤为学[4] 

机构地区：[1]重庆医科大学附属第一医院胃肠外科,重庆400016 [2]云南省第一人民医院普通外科,云南昆明650032 [3]重庆医科大学附属第一医院实验研究中心,重庆400016 [4]重庆医科大学基础医学院病理生理学教研室,重庆400016

出　　处：《中国生物制品学杂志》2013年第10期1445-1448,1453,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30972872)

摘　　要：目的探讨人表皮生长因子受体显性负性突变体(dominant negative epidermal growth factor receptor,DNEGFR)对人胃癌细胞体外生长能力的影响及其分子机制。方法用质粒pEGFPN1-DNEGFR转染人胃癌NCI-N87和SGC-7901细胞,并筛选稳定转染细胞株;采用MTT法检测人胃癌细胞体外生长能力,末端脱氧核苷酰基转移酶介导性dUTP切口末端标记[terminal deoxynucleotidyl transferase(TdT)mediated deoxyuridine triphosphate(dUTP)nick-end labeling,TUNEL]法检测细胞凋亡情况,ELISA法检测胞浆中活性caspase-3蛋白水平,Western blot法检测ser 9位点磷酸化糖原合成激酶-3β[phosphorylated glycogen synthase kinase-3 beta,pGSK-3β(ser 9)]蛋白表达水平。结果成功筛选出4株稳定转染细胞株;质粒pEGFPN1-DNEGFR分别转染的NCI-N87和SGC-790细胞体外生长能力均降低,凋亡指数(apoptotic index,AI)均升高,胞浆中活性caspase-3蛋白水平均升高,pGSK-3β(ser 9)蛋白水平均降低,与对应的空质粒转染组及未转染组相比,差异均有统计学意义(P<0.05)。结论 DNEGFR通过caspase相关的凋亡信号转导通路及糖原合成激酶-3β(GSK-3β)促凋亡作用诱导胃癌细胞凋亡,使其体外生长能力明显降低,为DNEGFR在胃癌生物治疗中的深入研究提供了部分理论依据,为胃癌的生物治疗提供了新思路。Objective To investigate the effect of dominant negative epidermal growth factor receptor （DNEGFR） on the growth of human gastric cancer cells as well as the relevant molecular mechanism. Methods Human gastric cancer NCI- N87 and SGC-7901 cells were transfected with plasmid pEGFPN1-DNEGFR, from which stably transfected cell strains were screened and determined for growth in vitro by MTI＂ assay, for apoptosis by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick-end labeling （TUNEL） assay, for active caspase-3 protein level in cytoplasm by ELISA, and for expression level of phosphorylated glycogen synthase kinase-3 beta at ser 9 [pGSK-3β （ser 9）] by West- ern blot. Results Four stably transfected human gastric cancer cell strains were screened. The growth abilities in vitro of NCI-N87 and SGC-790 cells transfected with pEGFPN1-DNEGFR decreased significantly, while the apoptotic indexes （AIs） increased, the active caspase-3 protein levels in cytoplasm increased, and pGSK-3β （ser 9） level decreased, which showed significant difference with those in the corresponding cells transfected with empty plasmid and those untransfected （P 〈 0. 05）. Conclusion DNEGFR induced the apoptosis of human gastric cancer ceils by caspase-mediated apoptosis signaling pathway and the pro-apoptotic effect of glycogen synthase kinase-3 beta （GSK-3β）, leading to the inhibition of cell growth in vitro, which provided a new idea for the biotherapy of gastric cancer.

关 键 词：人表皮生长因子受体 显性负性突变体 胃癌 细胞凋亡 

分 类 号：Q255[生物学—细胞生物学] R735.2[医药卫生—肿瘤]