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目的 :观察重组变构型人肿瘤坏死因子相关的凋亡诱导配体(recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand,rmh TRAIL)对人肺癌A549细胞株的凋亡诱导作用,验证与此凋亡相关的差异蛋白质表达谱。方法 :A549细胞经rmh TRAIL处理后,采用MTT法检测细胞增殖抑制率,FCM法检测细胞周期及细胞凋亡变化。采用液相色谱串联质谱(liquid chromatograph-tandem mass spectrometer,LC-MS/MS)鉴定技术对rmh TRAIL作用前后A549细胞的蛋白质组学进行对比分析,寻找差异表达明显的凋亡相关蛋白,然后用实时荧光定量PCR和蛋白质印迹法验证相关基因的表达水平。结果 :不同质量浓度的rmh TRAIL处理A549细胞24 h后,细胞增殖抑制率均显著提高(P值均<0.05),半数抑制浓度为(9.5±3.9)×105 ng/m L。5×105 ng/m L rmh TRAIL处理24 h可诱导A549细胞凋亡,早期凋亡率明显升高(P<0.01)。rmh TRAIL对细胞周期无明显影响。rmh TRAIL作用后,A549细胞中肿瘤坏死因子受体超家族成员10B(tumor necrosis factor receptor superfamily member 10B,TNFRSF10B)和TNFRSF10D表达水平分别下调至21.3%和31.8%,细胞FLICE抑制蛋白(cellular FLICEinhibitory protein,c-FLIP)表达水平上调至2.867倍。实时荧光定量PCR及蛋白质印迹法检测结果证实,用药后作为TNFRSF成员之一的小分子死亡受体5(death receptor-5,DR5)的m RNA和蛋白表达均明显下调(P值均<0.05),而c-FLIP m RNA和蛋白表达则明显上调(P值均<0.05)。结论 :rmh TRAIL可诱导A549细胞凋亡,其原因可能与DR5表达下调及c-FLIP表达上调有关。
AIM: To investigate the apoptosis-inducing effect of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmh TRAIL) on human lung cancer A549 cell line Apoptosis-related differential protein profiling. Methods: After A549 cells were treated with rmh TRAIL, the cell proliferation inhibition rate was detected by MTT assay. The cell cycle and apoptosis were detected by FCM. Using liquid chromatography-tandem mass spectrometer (LC-MS / MS) identification technology, we compared the proteomics of A549 cells before and after rmh TRAIL treatment, looked for differentially expressed apoptosis-related proteins, Fluorescent quantitative PCR and Western blot were used to verify the expression level of related genes. Results: After treated with rmh TRAIL for 24 h, the proliferation inhibition rates of A549 cells were significantly increased (P <0.05), and the median inhibitory concentration ((5 ± 3) × 10 5 ng / m L). A549 cells were treated with 5 × 105 ng / mL rmh TRAIL for 24 h, the apoptosis rate of A549 cells was significantly increased (P <0.01). rmh TRAIL had no significant effect on cell cycle. After rmh TRAIL treatment, the expression levels of TNFRSF10B 10B and TNFRSF10D in A549 cells were down-regulated to 21.3% and 31.8%, respectively. The levels of FLICE arrestin protein (FLICE) c-FLIP) was up-regulated to 2.867-fold. The results of real-time PCR and Western blotting confirmed that m RNA and protein expression of DRR receptor 5 (DR5), a member of TNFRSF, were significantly down-regulated after treatment (P <0.05) , While c-FLIP m RNA and protein expression were significantly increased (P values were <0.05). CONCLUSION: rmh TRAIL can induce the apoptosis of A549 cells, which may be related to the down-regulation of DR5 expression and the up-regulation of c-FLIP.