基因干预后胶质瘤A172细胞中HIF-1α和VEGF的表达

Expression of HIF-1α and VEGF in Glioma A172 Cells After Gene Intervention

目的观察体外基因干预后HIF-1α和VEGF表达的变化,从而探讨HIF-1α在胶质瘤血管新生调控中所起的作用。方法CoCl2化学缺氧法模拟肿瘤缺氧环境,反转录聚合酶链式反应(qPCR)、免疫荧光法、Westernblot法和ELISA双抗体夹心法比较基因干预HIF-1α前后,胶质瘤A172细胞相关mRNA和蛋白表达量变化。结果缺氧环境下HIF-1αmRNA表达未见明显变化,VEGFmRNA表达明显增多;缺氧培养8、16、24h,HIF-1α及VEGF蛋白表达依次增多。基因干预成功后缺氧培育24h,HIF-1α及VEGF蛋白表达明显减少,与未转染组比较,转染抑制率分别是86.3%和78.9%,VEGFmRNA表达抑制率是81.5%。结论siRNA干预抑制HIF-1α表达后VEGF表达明显减少。缺氧促使A172细胞HIF-1α在蛋白水平表达升高,HIF-1α通过转录激活VEGF促进胶质瘤缺氧状态下的血管新生。

Objective To observe the changes of hypoxia induction factor-a(HIF-α) and vascular endothelial growth factor(VEGF) expression after in vitro gene intervention, so as to explore the role of HIF-1α in the regulation of angiogenesis in glioma. Methods CoCl2 chemical hypoxia method simulated tumor hypoxia environment, and reverse transcription polymerase chain reaction(qPCR), immunofluorescence method,Western blot method and ELISA double antibody sandwich method were used to compare the expression levels of related mRNA and protein in glioma A172 cells before and after gene intervention of HIF-1α. Results HIF-1α mRNA expression was not significantly changed under hypoxia, and VEGF mRNA expression was significantly increased. The protein expressions of HIF-1α and VEGF were increased successively in hypoxic culture at 8, 16 and 24 hours. After successful gene intervention in hypoxia culture for 24 hours, HIF-1α and VEGF protein expressions were significantly reduced. Compared with the untransfected group, the inhibition rates of transfection were 86.3% and 78.9% respectively, and the inhibition rate of VEGF mRNA expression was 81.5%. Conclusion VEGF expression is significantly reduced after HIF-1α expression is inhibited by si RNA intervention. The protein expression of HIF-1α in A172 cells is increased in the hypoxic environment,and HIF-1α promotes angiogenesis in glioma under hypoxia through transcriptional activation of VEGF.