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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 effects of DNEGFR on the growth of human gastric cancer cells in vitro and its molecular mechanism. Methods Human gastric cancer NCI-N87 and SGC-7901 cells were transfected with plasmid pEGFPN1-DNEGFR, and the stable transfected cell lines were screened. The growth of human gastric cancer cells in vitro was detected by MTT assay. The terminal deoxynucleotidyl transferase-mediated dUTP nick Cell apoptosis was detected by terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) method. The level of active caspase-3 protein in cytoplasm was detected by ELISA, Phosphorylated glycogen synthase kinase-3 beta (pGSK-3β (ser 9)] protein expression levels. Results Four stable transfected cell lines were successfully screened. The ability of growth of NCI-N87 and SGC-790 cells transfected with pEGFPN1-DNEGFR plasmid was decreased, the apoptotic index (AI) The protein level of active caspase-3 increased and the protein level of pGSK-3β (ser 9) decreased. Compared with the corresponding empty plasmid transfected group and untransfected group, the difference was statistically significant (P <0.05). Conclusion DNEGFR induces the apoptosis of gastric cancer cells through the apoptosis-related signal transduction pathway of caspase and the pro-apoptotic effect of glycogen synthesis kinase-3β (GSK-3β), so that DNEGFR can significantly reduce the growth of gastric cancer cells in vitro. In-depth research provides some theoretical basis for the biological treatment of gastric cancer provides a new idea.