A549细胞条件培养液激活PI3K-Akt1信号通路促人脐静脉内皮细胞存活

Conditioned medium from lung carcinoma cell line A549 increases the viability of human umbilical vein endothelial cells by activating the PI3K-Akt1 pathway

目的:研究肺癌A549细胞条件培养液(conditioned medium,CM)对人脐静脉内皮细胞(human umbilical vein endo-thelial cells,HUVECs)生存活性和凋亡的影响,以及PI3K-Akt信号通路在其中的作用。方法:用制备获得的人肺癌A549细胞CM培养HUVECs;XTT法检测HUVECs活性;Hoechst33258染色,荧光显微镜观察细胞凋亡的形态学改变;FCM法检测细胞的凋亡率;Western印迹法检测Akt总蛋白和磷酸化Akt蛋白表达。分别用特异性PI3K抑制剂wortmannin(WT)和针对Akt1基因的siRNA(siAkt1)转染HUVECs,RT-PCR法检测Akt亚型mRNA的表达,并观察上述各指标的变化。结果:在CM刺激后24h,HUVECs活性明显增强(P=0.037),凋亡率下降(P=0.001)。CM呈时间依赖性激活HUVECsAkt磷酸化,CM处理后15min即显示磷酸化Akt水平增高,30min达高峰,此后呈下降趋势。WT或siAkt1转染均能阻断前述效应。结论:肺癌A549细胞CM经PI3K-Akt途径促进HUVECs存活并抑制其凋亡,其中Akt1起关键作用。

Objective：To study the effects of the conditioned medium （CM） from human lung adenocarcinoma cell line A549 on the viability and apoptosis of human umbilical vein endothelial cells （HUVECs） and the role of PI3K-Akt signaling pathway in the process. Methods：HUVECs were cultured with CM of A549 cells. Cell viability was detected by XTT assay. The morphological changes of HUVECs were analyzed by Hoechst 33258 staining and fluorescence microscopy. The apoptosis was detected by flow cytometry. Expression levels of total Akt and phosphorylated Akt were assessed by Western blotting. PI3K inhibitors wortmannin（WT）and Akt1 siRNA（siAkt1）were used to block PI3K-Akt signaling pathway. The mRNA transcription of Akt subtype was determined by RT-PCR.Results：A549 CM significantly increased cell viability after 24 h treatment （P=0.037） and inhibited apoptosis （P=0.001） of HUVECs. CM time-dependently activated phosphorylation of Akt. Akt was phosphorylated at 15 min after CM treatment and reached the peak at 30 min and then tended to decline. Both WT and siAkt1 blocked the effects of CM. Conclusion：The CM of A549 cells increased the survival and inhibit the apoptosis of HUVECs. Akt1 played a significant role in the process.