【摘要】 本研究探讨硼替佐米对内皮细胞株HMEC-1 VEGF基因表达的影响,并检测低氧诱导因子-1α(HIF-1α)转录调节活性和膜联蛋白A2(Annexin A2)表达强度的变化以分析VEGF基因表达变化可能的机制。用细胞计数试剂盒CCK-8检测细胞的相对增殖活力;采用实时定量PCR检测VEGF基因表达强度,RT-PCR检测碳酸酐酶IX(CAIX)的基因表达强度,用实时定量PCR和Western blot分别检测Annexin A2在基因和蛋白水平的变化。结果表明:2.5、5.0、10nmol/L硼替佐米作用12小时,内皮细胞株HMEC-1VEGF基因表达强度分别为:0.730±0.106、0.673±0.153、0.767±0.090(0nmol/L时为1.0)(p<0.05);2.5、5.0nmol/L药物未明显抑制细胞的增殖活力(p>0.05),10nmol/L时细胞增殖则明显被抑制(p=0.024),不同浓度硼替佐米处理后,CAIX基因表达强度均明显降低,HIF-1α转录调节活性受到抑制;Annexin A2蛋白的表达也明显受抑。结论:低剂量的硼替佐米对蛋白酶体活性无明显抑制作用;硼替佐米可能通过调节内皮细胞HIF-1α的活性和Annexin A2蛋白的表达下调VEGF基因的表达水平。更多还原

【Abstract】 This study was aimed to investigate the effects of bortezomib on VEGF gene expression of endothelial cell line HMEC-1, and to determine the changes of the transcriptional regulation activity of hypoxia-inducible factor 1(HIF-1α) and expression intensity of Annexin A2, so as to analyze the possible mechanisms of the above expression of VEGF gene. Expression intensity of VEGF gene was determined by real-time quantitative PCR; the relative proliferation activity of cells was assayed by cell count kit CCK-8; the expression intensity of carbonic anhydrase IX (CA IX) gene was detected by RT-PCR; expression of Annexin A2 at gene and protein levels were determined by real-time quantitative PCR and Western blot respectively. The results showed that after being treated by bortezomib with 2.5, 5.0, 10 nmol/L for 12 hours, the expression intensitiy of VEGF gene of endothelial cell line HMEC-1 was as follows: 0.730±0.106, 0.673±0.153, 0.767±0.090(as 1.0 was made in 0 nmol/L)(p<0.05);the proliferation activity of cells was not significantly suppressed by bortezomib in 2.5, 5.0 nmol/L(p>0.05), while that was significantly suppressed by bortezomib of 10 nmol/L (p=0.024), The results from RT-PCR showed that expression intensity of CA IX gene was conspicuously down-regulated by bortezomib in different concentrations, which suggested that the transcriptional regulation activity of HIF-1α was inhibited by bortezomib. And down-regulated expression of Annexin A2 protein by bortezomib in different concentrations was confirmed by real-time quantitative PCR and Western blot. It is concluded that low doses of bortezomib has no significant inhibition effect on the activity of proteasome. Bortezomib may down-regulate the expression of VEGF gene of endothelial cell through regulating the activity of HIF-1α and the expression of Annexin A2.更多还原