低氧处理不同时间对人肺腺癌A549细胞增殖的影响及相应缺氧模型探讨

Effect of Different Time of Hypoxia Treatment on the Proliferation of Human Lung Adenocarcinoma A549 Cells and the Associated Experimental Study of Hypoxic Model

目的:观察低氧处理不同时间对人肺腺癌A549细胞增殖的影响,探讨合理的人肺腺癌细胞株A549体外模拟缺氧时间。方法:将人肺腺癌细胞A549细胞株在低氧环境下分别培养12 h、24 h、48 h、72 h,设置常氧对照组,通过CCK8法测定A549细胞存活率,RT-PCR和免疫印迹分别检测细胞缺氧诱导因子-1α(hypoxia-inducible factor-1α, HIF-1α)和血管内皮生长因子(vascularendothelial growth factor, VEGF)mRNA及蛋白的表达。结果:低氧24 h组A549细胞存活率最高,低氧48 h、72 h组A549细胞存活率呈时间依赖性明显下降(P<0.001)。自低氧12 h起,A549细胞HIF-1αmRNA和VEGFmRNA的表达开始随低氧时间延长而显著增加(P均<0.001);HIF-1α和VEGF蛋白表达自24 h开始随低氧时间延长而显著增加(P均<0.001)。结论:低氧诱导的A549细胞存活率呈时间依赖性降低,而HIF-1α、VEGF表达呈时间依赖性增高,人肺癌细胞株A549缺氧模型最适时间为24 h。

ABSTRCT Objective: To observe the effect of different time of hypoxia treatment on the proliferation of human lung adenocarcinoma A549 cells. To explore the optimal time for the hypoxic model in vitro for human lung adenocarcinoma cell line A549.Methods: Human lung adenocarcinoma A549 cells were cultured under hypoxic environment for 12 h, 24 h, 48 h and 72 h respectively and set up a normoxic group as contrast. The proliferation of A549 cells was detected by CCK8 assay. The transcriptional mR NA and protein levels of hypoxia-inducible factor-1α(HIF-1α) and vascular endothelial growth factor(VEGF) were examined by RT-PCR and western blot. Results: The survival rate of the 24 h group was the highest, and the cells’ proliferation of 48 h and 72 h group significantly decreased in a time-dependent manner(P<0.001). The mR NA expression of HIF-1α and VEGF began to increase significantly from 12 h while the protein expression rose significantly from 24 h. After the above time points the mR NA and protein expression both increased with the prolongation of hypoxic time(P <0.001). Conclusions: The viability of hypoxia-induced A549 cells decreased in a time-dependent manner while the expression of HIF-1α and VEGF increased with the extension of hypoxic time. The optimal time for A549 hypoxic model in our study was 24 h.