论文部分内容阅读
目的:通过顺铂诱导人肺腺癌细胞A549的凋亡,研究X染色体连锁凋亡抑制蛋白相关因子1(X-linked inhibitor of apoptosis protein associated factor-1,XAF1)基因在A549细胞中的表达情况,初步探讨该基因在凋亡中的作用。方法:采用4μg/mL浓度的顺铂处理A549细胞,MTT法检测细胞的增殖抑制率;AnnexinⅤ/7-AAD检测A549细胞凋亡情况;RT-PCR和蛋白质印迹法分别检测A549凋亡后XAF1mRNA和蛋白的表达;Caspase-3试剂盒检测A549细胞凋亡后Caspase-3的活性。结果:用4μg/mL浓度顺铂处理A549细胞2、12和24h后,A549细胞增殖抑制率分别为(3.40±0.65)%、(8.31±0.73)%和(14.72±0.24)%,与对照组(1.03±0.28)%相比均明显增高,P值分别为0.004、0.000和0.000,细胞增殖抑制率随时间增加而增强;A549细胞凋亡率分别为(4.35±0.95)%、(9.69±2.60)%和(22.35±1.24)%,与对照组(1.39±0.21)%相比均明显增加,P值分别为0.006、0.005和0.000,细胞凋亡率随顺铂作用时间延长而增加;实验组XAF1mRNA表达水平分别为(0.199±0.029)、(0.654±0.093)和(1.216±0.101),对照组为(0.091±0.020),XAF1mRNA表达水平随作用时间的延长呈增加趋势,P值分别为0.006、0.001和0.000;实验组XAF1蛋白表达水平分别为(0.322±0.041)、(0.508±0.014)和(0.901±0.014),对照组为(0.124±0.007),XAF1蛋白表达水平随作用时间的延长呈增加趋势,P值分别为0.001、0.000和0.000;Caspase-3活性(A405nm)分别为(34.745±3.781)、(69.524±3.096)和(94.787±5.429),对照组为(21.914±1.962),Caspase-3活性随顺铂作用时间的延长而增加。结论:顺铂诱导A549细胞凋亡可以引起XAF1表达增加,且随细胞凋亡水平的提高而增加,提示XAF1可能参与顺铂诱导人肺腺癌A549细胞的凋亡过程。
OBJECTIVE: To investigate the expression of X-linked inhibitor of apoptosis protein associated factor-1 (XAF1) in human lung adenocarcinoma A549 cells induced by cisplatin , Preliminary study of the role of the gene in apoptosis. Methods: A549 cells were treated with cisplatin (4μg / mL) and the proliferation of A549 cells were detected by MTT assay. The apoptosis of A549 cells was detected by AnnexinⅤ / 7-AAD. The expressions of XAF1 mRNA and protein were detected by RT- Caspase-3 kit was used to detect the activity of Caspase-3 after A549 cell apoptosis. Results: After treated with 4μg / mL cisplatin for 2, 12 and 24 hours, the proliferation inhibition rates of A549 cells were (3.40 ± 0.65)%, (8.31 ± 0.73)% and (14.72 ± 0.24)%, respectively. (1.03 ± 0.28)%, the P values were 0.004, 0.000 and 0.000 respectively, and the cell proliferation inhibition rate increased with time increasing. The apoptosis rates of A549 cells were (4.35 ± 0.95)% and (9.69 ± 2.60 ) And (22.35 ± 1.24)% respectively, which were significantly increased compared with the control group (1.39 ± 0.21)%, the P values were 0.006,0.005 and 0.000 respectively. The apoptotic rate increased with the prolongation of cisplatin treatment. The expression levels of XAF1mRNA were (0.199 ± 0.029), (0.654 ± 0.093) and (1.216 ± 0.101) in the control group and (0.091 ± 0.020) in the control group, respectively. The expression of XAF1mRNA increased with the prolongation of time, P values were 0.006, 0.001 and 0.000 respectively. The expression of XAF1 protein in the experimental group was (0.322 ± 0.041) and (0.508 ± 0.014) and (0.901 ± 0.014) respectively, while the control group was (0.124 ± 0.007). The XAF1 protein expression level increased with the extension of time (P = 0.001,0.000 and 0.000). Caspase-3 activity (A405nm) were (34.745 ± 3.781), (69.524 ± 3.096) and (94.787 ± 5.429) .914 ± 1.962). The activity of Caspase-3 increased with the prolongation of the action of cisplatin. CONCLUSION: Apoptosis of A549 cells induced by cisplatin can increase the expression of XAF1 and increase with the increase of cell apoptosis, suggesting that XAF1 may be involved in cisplatin-induced apoptosis of human lung adenocarcinoma A549 cells.