摘要
目的研究肺癌A549细胞条件培养基对血管内皮细胞凋亡的影响及机制。方法以5%胎牛血清(fetal bovine serum,FBS)为对照,用人肺癌A549细胞株条件培养基孵育脐静脉内皮细胞(human umbilical veins endothelial cells,HUVECs),Annexin V-FITC试剂盒染色,流式细胞仪进行细胞凋亡定量。设计针对蛋白激酶B(protein kinase B,Akt1)基因mRNA的siRNA(siAkt1)和非特异性对照序列(siSCR)转染HUVECs,用RT-PCR技术检测Akt亚型mRNA的表达,用Western blotting技术检测Akt总蛋白表达,并观察细胞凋亡的变化。结果 A549条件培养基明显抑制了HUVECs的凋亡(凋亡率3.03%,显著低于对照组的12.69%,P<0.05)。筛选出的si Akt1能显著抑制HUVECs的Akt1 mRNA和Akt总蛋白表达(分别下调81%和62%),转染si Akt1后的HUVECs再用A549条件培养基孵育凋亡明显增强(与siSCR组相比P<0.01)。结论肺癌A549细胞条件培养基经Akt1途径抑制血管内皮细胞凋亡,这将为肺癌血管生成的机制研究和干预治疗提供新的分子靶点。
Objective To investigate the effect and mechanism of human lung carcinoma A549-derived conditioned medium （A549CM） on apoptosis of human umbilical veins endothelial cells （HUVECs）.Methods HUVECs were treated with the A549CM,and cultured in 5% fetal bovine serum （FBS） as control.The apoptosis of HUVECs was assessed by Annexin-V-FITC labeling and fluorescence-activated cell sorting analysis.The small interfering RNA sequence specifically targeting protein kinase B Akt1 （named as siAkt1） and the scramble siRNA sequence ＂siSCR＂ （served as control） were constructed and chemically synthesized,and transfected into HUVECs with LipofectamineTM 2000.The expression of Akt1-mRNA was examined by RT-PCR,and the Akt protein expression level was checked by Western blotting analysis.The apoptosis of HUVECs was assessed.Results Compared with the control group,the A549CM significantly inhibited the apoptosis of HUVECs （3.03% vs 12.69%）,siAkt1 significantly depressed the Akt1-mRNA （81%）and Akt protein （62%） expression level of HUVECs,and the apoptosis of siAkt1 and A549CM treated HUVECs increased significantly （P0.01）.Conclusion Lung carcinoma A549-derived conditioned medium can depress the apoptosis of HUVECs by activating Akt1,which may provide a new molecular target for research on vasculogenesis of lung cancer and intervention therapy.
出处
《华南国防医学杂志》
CAS
2010年第4期272-274,277,共4页
Military Medical Journal of South China
基金
湖北省十堰市科技局重点项目(2005ZD017
2006ZD09)
关键词
肺癌
内皮细胞
凋亡
蛋白激酶B
Lung cancer
Endothelial cells
Apoptosis
Protein kinase B
