PDCD5蛋白联合顺铂对A549细胞凋亡影响研究

Effect of PDCD5 protein combined cisplatin on apotosis in A549 cells

目的通过重组人程序性细胞死亡分子5(rhPDCD5)蛋白与顺铂单独或联合作用于肺腺癌A549细胞,探讨rhPDCD5联合顺铂对细胞凋亡的影响,并探讨其可能的作用机制。方法采用MTT法分别检测不同浓度的rhPDCD5蛋白(5、10、15和30mg/L)、不同浓度的顺铂(2.5、5、10、20和40mg/L)及20mg/L顺铂、15mg/L rhPDCD5蛋白单独/联合作用24h后对人肺腺癌A549细胞的增殖抑制率;采用流式细胞仪检测对照组、rhPDCCD5组(15mg/L)、顺铂组(20mg/L)及联合用药组细胞凋亡;蛋白质印迹法测检测各组间细胞中Caspase-3表达。结果 MTT结果显示,与对照组相比,不同浓度rhPDCD5蛋白作用于A549细胞,对细胞增殖抑制率的作用不明显,F=0.114,P=0.949;随着顺铂浓度的增高,抑制作用逐步增强,药物剂量与细胞增殖抑制率呈正相关,F=88.564,P<0.01;联合用药组较单药组细胞表现出更强的抑制作用,F=1 258.284,P<0.01。流式细胞仪检测结果显示,对照组凋亡率为(7.77±0.15)%,rhPDCD5组为(9.37±0.31)%,顺铂组为(23.13±0.35)%,rhPDCD5+顺铂组为(31.33±0.45)%,组间比较差异有统计学意义,F=3 458.747,P<0.01。蛋白质印迹法结果显示,单用rhPDCD5组较对照组cleaved Caspase-3表达上调不明显,顺铂组及联合用药组cleaved Caspase-3表达上调,且联合用药组表达上调最明显,F=92.734,P<0.01。结论 rhPDCD5蛋白单独应用对A549细胞的增殖及凋亡作用不明显;rhPDCD5蛋白可明显增加顺铂抑制A549细胞增殖、诱导细胞凋亡的作用,其机制可能是通过激活Caspase-3信号通路促进顺铂诱导A549细胞凋亡,增加顺铂的化疗敏感性,在治疗肺腺癌中联合用药可能会具有较大的应用潜力。

OBJECTIVE To investigate the effect of rhPDCD5 combined with cisplatin on apoptosis of lung adeno- carcinoma and its possible mechanism. METHODS The proliferation inhibition rates of the A549 cells that were treated with various concentrations of rhPDCD5 and cisplatin on 24 h were detected by MTT assay. The cells were divided into 4 groups., normal control group, rhPDCD5 group, cisplatin group, rhPDCD5 and cisplatin group. After 24 h treatment, the effects of drugs on growth were detected by MTT assay, the apoptoses were measured by flow cytometric,Caspase-3 ac tivity of A549 cells was evaluated by Western blotting. RESULTS The proliferation inhibition rates of the cancer cells treated with different concentrations of rhPDCD5 were similar to those of controls （F = 0. 114, P = 0. 949）, while those cells had treated with different concentrations of cisplatin（F= 88. 564, P〈0.01）. Compared to the single treatment, the combined treatment with rhPDCD5 and cisplatin had a higher inhibiting cell proliferation（F= 1 258. 284, P〈0.01）. Apop- totic ratio of control group, rhPDCD5 group, cisplatin group, rhPDCD5 and cisplatin group were （7.77 ± 0.15） %, （9.37 ± 0.31）%,（23.13±0.35） % , （31. 33±0. 45）%,there were differences among these four groups（F=3 458. 747,P〈0.01）. Western blotting results showed that cleaved Caspase-3 of treated with cisplatin,rhPDCD5 and cisplatin were significantly higher than that in control group and only treated with rhPDCDS, with the highest raising in combination group （F = 92. 734,P〈0.01）. CONCLUSION RhPDCD5 protein does not work alone to A549 cells; rhPDCD5 protein may increase activation of Caspase-3 to play its role in promoting apoptosis and strengthen the chemotherapy sensitivity, combined treatment has a great potential in the treatment of non-small cell lung cancer.