摘要
目的研究血管内皮细胞生长因子(VEGF)对乳腺癌细胞株MCF-7的凋亡及凋亡相关基因survivin和bcl-2表达的影响,探讨VEGF自分泌作用抑制肿瘤细胞凋亡的机制。方法构建VEGF_(165)真核表达质粒,采用脂质体转染VEGF_(165) cDNAMCF-7细胞,通过RT-PCR及ELISA方法鉴定转染克隆MCF-7／hVEGF_(165)细胞中VEGF mRNA及蛋白的表达,Westernblot方法比较MCD-7／hVEGF_(165)及MCF-7、MCF-7／pcDNA3细胞中survivin、bcl-2蛋白表达,流式细胞仪分析细胞凋亡和细胞周期。结果 MCF-7／hVEGF_(165)细胞VEGF mRNA表达量增加,细胞培养上清中VEGF蛋白浓度为(354．50±31．93)ng／L,高于MCF-7细胞的(178．54±16．52)ng／L和MCF-7／pcDNA细胞的(190．75±13．32)ng／L(P d0．01)。与MCF-7、MCD-7／pcDNA_3组相比较．MCF-7／hVEGF_(165)细胞凋亡百分数显著下降(P<0．05),分别为(2．47±0．51)％(MCF-7／hVEGF_(165)),(7．74±1．56)％(MCF-7／pcDNA3)和(7．35±0．33)％(MCF-7)；survivin和bel-2蛋白表达增高了近3倍,但各组细胞周期变化不明显(P>0．05)。结论 VEGF诱导凋亡抑制基因suvivin和bel-2高表达,可能在自分泌VEGF抑制MCD-7凋亡中发挥重要作用。
Objective To explore the effect of autocrine vascular endothelial growth factor（VEGF） on the apoptosis of MCF-7 and its possible mechanisms. Methods The expressive plasmid pcDNA3/hVEGF165 was constructed and transfected via lipofictamine into MCF-7 cells （MCF-7/hVEGF165） . Reverse transcription-polymerase chain reaction （RT-PCR） was used for detecting VEGF mRNA in the transfected cells. VEGF concentrations in the cell culture supernatant were determined by enzyme-linked immunosorbent assay （ELISA）. The expressions of survivin and bcl-2 in transfected cells were detected by western blot . Flow cytometry was employed to observe the effect of VEGF165 cDNA transfection on the apoptosis and cell cycle of MCF-7. Results The levels of VEGF mRNA expression by MCF-7/hVEGF165 cells were higher than MCF-7/pcDNA3 and MCF-7,with the mean VEGF quantity in the supernatant of MCF-7/hVEGF165 cell culture being（354.50 ± 31.93）ng/L, which was about 2-fold higer than in the supernatant of MCF-7/pcDNA3 and MCF-7 cells（ P 〈0.01 ） . The expressions of survivin and bcl-2 in MCF-7/ hVEGF165 cells were significantly higer than other two groups. Less apoptosis cells were identified in MCF-7/hVEGF165 （2.47 ± 0.51 ） % cells than in MCF-7/pcDNA3 （7.74 ± 1.56） % and MCF-7 （7.35 ±0.33） % cells in the same culture condition. There was no alteration of cell cycle in three groups. Conclusion Our results have demonstrated that autocrine VEGF increases survivin and bcl-2 expression by human breast cancinoma cells, which may be play an important role in the mechanisms of the factor that autocrine VEGF inhibits apoptosis in human breast cancinoma cells.
出处
《肿瘤》
CAS
CSCD
北大核心
2005年第6期525-529,共5页
Tumor
基金
国家自然科学基金(编号:30470497)
上海市科委重点基金资助项目(编号:02DJ04137)
