摘要目的 观察顺铂(DDP)作用下肺癌A549细胞系的细胞周期和凋亡的动力学特点,以及灭活细胞周期检测点激酶1(Chk1)和细胞周期检测点激酶2(Chk2)基因对DDP诱导的肺癌细胞凋亡的影响.方法 优化转染条件后,将肺癌A549细胞分为8组进行转染,分别为正常组(未经任何处理的A549细胞)、对照组(A549细胞接受10 μmol/L的DDP作用12 h)、转染Chk1正义寡核苷酸链(sODN)组、转染Chk1反义寡核苷酸链(AsODN)组、转染Chk2 sODN组、转染Chk2 AsODN组、联合转染Chk1和Chk2 sODN组以及联合转染Chk1和Chk2 AsODN组,其中后6组转染24 h后再以10 μmol/L的DDP处理12 h.采用逆转录聚合酶链反应(RT-PCR)和Western blot法,检测转染Chk1或Chk2 sODN、AsODN后,A549细胞中Chk1或Chk2 mRNA和蛋白的表达.采用流式细胞仪Annexin V-FITC法和Sub-G1法,检测转染Chk1和(或)Chk2 sODN、AsODN后,DDP作用下A549细胞的细胞周期和凋亡变化.结果 10μmol/L的DDP作用12 h后,非同步化处理的A549细胞出现了明显的S期阻滞现象.转染24 h后,Chk1或Chk2 AsODN可明显抑制A549细胞中Chk1 mRNA和蛋白或Chk2 mRNA和蛋白的表达.与单独转染Chk1或Chk2 sODN组相比,单独转染Chk1或Chk2 AsODN组DDP诱导下A549细胞的凋亡率约提高了1～2倍(P＜0.05);但联合转染Chk1和Chk2 AsODN组与单独转染Chk1 AsODN或Chk2 AsODN组相比,A549细胞凋亡率的差异无统计学意义(P=0.521和P=0.339).结论 灭活Chk1和Chk2基因引起化疗增敏的机制可能是通过解除S期阻滞这种肿瘤细胞的自我保护机制实现的,Chk1和Chk2基因可作为肺癌化疗药物增敏治疗的有效靶点.

abstractsObjective To investigate the changes in cell cycle induced by cisplatin(DDP)and the effect of antisense oligonucleotide(AsODN)targeting Chk1/2 on DDP-induced apoptosis in lung cancer cell line A549 cells.Methods The characteristics of cell cycle and apoptosis induced by DDP were detected by flow cytometry using SubG1 method.Chk1/2 mRNA and protein expression were assayed by RT-PCR and Western blot under best conchtion of transfection of ABODN targeting Chk1/2 by lipofection.Apoptosis of A549 cells induced by DDP was determined by flow cytometry using AnnexinV-FITC staining after transfection of Chk1/2 AsODN.Results Asynchronized A549 cells were treated with 10 μmol/L DDP,and significant S-phase arrest was observed at 12 h later.Transfection with antisense oligonucleotide targeting Chk1/2 inhibited the Chk1/2 expression at both mRNA and protein levels.Either Chkl or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptesis by 100%-200%.compared with that in the sODN control(P＜0.05),but combined use of Chkl-and Chk2-specific AsODN did not show synergistic effects as compared witII that induced by treatment with Chk1-or Chk2-specific AsODN alone(P＞0.05).Conclusion Chk1 and Chk2 may be regarded as effective targets of chemotherapy for lung cancer.Silencing the key effector Chk1 and Chk2 genes may significantly increase the chemosensitivity of lung cancer cells.