PTEN基因转染对白血病细胞VEGF调控作用的影响

Regulatory effects of PTEN gene transfection on vascular endothelial growth factor(VEGF) in leukemia cells

目的:探讨在白血病细胞中与张力蛋白同源的10号染色体缺失的磷酸酶基因(phosphatase and tensin hemology dele-ted on chromosome ten gene,PTEN)对血管内皮生长因子(vascular endothelial growth factor,VEGF)及其受体1(VEGF receptor 1,VEGFR1)调控作用的影响。方法:将携带有野生型PTEN及绿色荧光蛋白(green fluorescent protein,GFP)基因的腺病毒(Ad-PTEN-GFP)及空载体腺病毒(Ad-GFP)转染人慢性粒细胞白血病急变细胞株K562,实时荧光定量PCR法检测不同转染组细胞中PTEN、VEGF和VEGF1 mRNA表达水平,Western印迹法检测PTEN、VEGF、Akt和磷酸化Akt蛋白的表达水平,并采用Transwell小室侵袭实验检测不同转染组细胞的侵袭性。通过MTT实验及FCM法检测PTEN基因对脐静脉内皮细胞株ECV304增殖和凋亡的影响。通过鸡胚尿囊膜(chick chorioallantoic membrane,CAM)体内血管生长实验检测PTEN基因对鸡胚血管生成的影响。结果:与空载体腺病毒(Ad-GFP)相比,Ad-PTEN-GFP转染人白血病细胞K562后,VEGF及其受体的表达被明显抑制,并呈剂量依赖性负相关;同时,Ad-PTEN-GFP转染后K562细胞侵袭能力明显减弱。PTEN基因转染能够抑制血管内皮细胞ECV304增殖,并促进其细胞凋亡,细胞周期阻滞在S期。PTEN基因转染可明显抑制CAM血管生长。结论:肿瘤抑制基因PTEN能够抑制内皮细胞增殖和白血病细胞侵袭,其作用机制可能是负调控白血病细胞VEGF表达以及抑制肿瘤血管新生。

Objective:To investigate the effects of wild type tumor-suppressing gene PTEN(phosphatase and tensin hemology deleted on chromosome ten gene) on vascular endothelial growth factor(VEGF) and VEGF receptor 1(VEGFR1) in leukemia cells and elucidate the mechanism.Methods:The recombinant adenovirus containing wild type PTEN and green fluorescent protein(GFP)(Ad-PTEN-GFP) or empty vector(Ad-GFP) was transfected into K562 leukemia cells.PTEN,VEGF,and VEGFR1 mRNA expression levels were detected using quantitative PCR and the protein expression levels were detected using Western blotting.Transwell invasion test was used to examine the invasion ability of the leukemia cells.Human umbilical vein endothelial cells ECV304 were also transfected with or without PTEN gene.Then the proliferation and apoptosis were measured using MTT assay and flow cytometry(FCM),res-pectively.Chick chorioallantoic membrane(CAM) test was used to determine the effect of PTEN gene transfection on angiogenesis.Results:The expressions of VEGF and VEGFR1 were significantly inhibited after transfection of Ad-PTEN-GFP compared with the control group transfected with empty Ad-GFP.The inhibitory effects were in a dose-dependent manner.The invasion capability of K562 cells was markedly weakened after transfection of Ad-PTEN-GFP.PTEN gene transfection inhibited the proliferation and induced apoptosis of vascular endothelial ECV304 cells.The cells were arrested at S phase.CAM test showed that PTEN gene inhibited the angiogenesis in vivo.Conclusion:Tumor suppressor gene PTEN inhibited the growth of endothelial cells and the invasion of leukemia cells.The action mechanism may be related with down-regulation VEGF expression and angiogenesis of leukemia cells.