摘要
目的:研究尼美舒利(NIM)与表皮生长因子(EGF)受体沉默联合应用对胶质瘤U251细胞作用及机制.方法:不同浓度的尼美舒利(0、25、50、100、200、400μmol/L)与靶向EGF受体的小分子RNA(siRNA-EGFR)联合应用培养U251胶质瘤细胞,ELISA试剂盒检测培养上清中血管内皮生长因子(VEGF)水平,MTT法检测细胞的增殖并计算抑制率,免疫印迹检测细胞增殖、凋亡和血管形成调控相关蛋白,流式细胞仪测定细胞周期和凋亡.结果:siRNA-EGFR浓度小于2μg/ml联合NIM(25~400 μmol/L)处理U251细胞,其生长抑制率与NIM的浓度依赖性增高,流式检测阻滞U251细胞于G2/M期及诱导细胞凋亡,蛋白分析表明,当沉默U251细胞EGF受体时,Bax蛋白表达随尼美舒利浓度增加表达上调,而bcl-2、细胞增殖核抗原(PCNA)和促血管生成素-2(ANG-2)蛋白表达明显下调,促血管生成素-1(ANG1)表达变化不明显;siRNA-EGFR与25 μumol/L的尼美舒利处理U251胶质瘤细胞12 h后,流式细胞仪检测出现典型的凋亡亚二倍体峰,并且随尼美舒利处理时间的延长,凋亡峰增加,随处理尼美舒利剂量的增加而呈现增加趋势,各组间差异具有统计学意义.结论:当siRNA-EGFR浓度为2μg/ml以下和作用时间在48 h之前,EGF受体沉默联合尼美舒利能发挥协同作用,其机制可能与Bax蛋白表达上调和bcl-2、PCNA、VEGF、ANG-2蛋白表达明显下调有关.
Objective: To investigate the proliferation-inhibiting and induce the apoptosis by combination with nimesulide (NIM) and silencing epidermal growth factor (EGF) receptor on glioma cells U251. Methods: Human U251 glioma cells were incubated by combination with NIM (0,25,50,100,200,400 tzmol/L) and inhibiting RNA targeted EGF receptor (siRNA EGFR) for 12, 24, 48 and 72 h. The vascular endothelial growth factor (VEGF) level in the culture supernatant was quantitated by ELISA assay, and the growth inhibition rate of U251 was investigated by MTT assay. The cell cycle and apoptosis were tested by flow cytometry; The cell proliferation, apoptosis and angiopoiesis related proteins were tested by Western blotting. Results: The combination of NIM and siRNA-EGFR inhibited the growth of U251 cells more effectively in a concentration-time dependent way than did siRNA-EGFR or COX-2 inhibitor. The combination with siRNA-EGFR and NIM showed a dose-dependent increase in G2/M phase arrest and apoptosis, and up-regulated the expression of Bax and down- regulated the expression of bcl-2, VEGF, ANG-2. Conclusion: Cell growth inhibition rates were significantly increased by the combination of the NIM and siRNA-EGFR played additive action, mechanism of which is involved in the up-regulation of the expression of Bax, and down-regulation of the expression of bcL2, VEGF and ANG-2.
出处
《解剖学杂志》
CAS
CSCD
北大核心
2013年第4期794-799,共6页
Chinese Journal of Anatomy
基金
湖北省自然科学基金(301131485)