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探讨沙蟾毒精(arenobufagin)对体内外血管生成的抑制作用。采用MTT法检测沙蟾毒精对人鼻咽癌细胞(CNE-2)、人喉癌细胞(Hep2)、人神经母细胞瘤(SH-SY5Y)、人结肠癌细胞(LOVO)、人前列腺癌细胞(PC-3及DU145)和人脐静脉内皮细胞(HUVECs)的细胞毒性及运用光学显微镜观察细胞形态的变化,发现沙蟾毒精剂量依赖性抑制CNE-2、Hep2、SH-SY5Y、LOVO、PC-3、DU145和HUVECs增殖,且在对人癌细胞亚细胞毒浓度下能够有效抑制HUVECs的增殖。采用鸡胚尿囊膜(chick embryo chorioallantoic membrane,CAM)模型观察新生血管的生成,结果发现沙蟾毒精作用24 h后,尿囊膜新生血管生成明显减少。采用细胞周期分析法观察到沙蟾毒精阻滞HUVECs于G2/M期,且随着浓度增加,细胞出现sub-G1凋亡峰。运用流式细胞术检测沙蟾毒精作用HUVECs后的凋亡现象以及线粒体膜电位的变化,确证沙蟾毒精呈时间和剂量依赖性诱导HUVECs凋亡,同时检测到线粒体膜电位下降。Western blotting检测发现,沙蟾毒精作用HUVECs后,凋亡标志分子PARP被切割。上述研究表明,沙蟾毒精具有明显的体内外抑制血管生成作用,其抑制作用可能与诱导血管内皮细胞周期阻滞及凋亡有关。
To investigate the inhibitory effect of arenobufagin on angiogenesis in vitro and in vivo. The proliferation of human nasopharyngeal carcinoma cells (CNE-2), human laryngeal carcinoma cells (Hep2), human neuroblastoma cells (SH-SY5Y), human colon cancer cells (LOVO), human prostate cancer Cytotoxicity of cells (PC-3 and DU145) and human umbilical vein endothelial cells (HUVECs) and the morphological changes of cells were observed by light microscopy. It was found that ADP inhibited CNE-2, Hep2, SH- SY5Y and LOVO , PC-3, DU145 and HUVECs proliferation, and can effectively inhibit the proliferation of HUVECs in the sub-cytotoxic concentration of human cancer cells. The formation of neovascularization was observed by the model of chick embryo chorioallantoic membrane (CAM). The results showed that the neovascularization of alveolar membrane was significantly reduced after 24 hours of lasagin treatment. The apoptosis of HUVECs was observed at G2 / M phase by cell cycle analysis. The sub-G1 apoptotic peak appeared with the increase of concentration. Flow cytometry was used to detect the apoptosis of HUVECs induced by SARS and the changes of mitochondrial membrane potential. The apoptosis of HUVECs was induced by time and dose-dependently, and the decrease of mitochondrial membrane potential was also detected. Western blotting showed that apoptotic marker PARP was cleaved after sabatumbaroxene treatment of HUVECs. The above studies show that safin toxin has a significant inhibitory effect on angiogenesis in vitro and in vivo, and its inhibitory effect may be related to the induction of vascular endothelial cell cycle arrest and apoptosis.