中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2009年
11期
804-809
,共6页
叶飞%谢大兴%卢运萍%高庆蕾
葉飛%謝大興%盧運萍%高慶蕾
협비%사대흥%로운평%고경뢰
化学疗法%Chk1%Chk2%细胞周期%凋亡
化學療法%Chk1%Chk2%細胞週期%凋亡
화학요법%Chk1%Chk2%세포주기%조망
Cliemothempy%Chk1%chk2%Cell cycle%Apoptosis
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.
目的 觀察順鉑(DDP)作用下肺癌A549細胞繫的細胞週期和凋亡的動力學特點,以及滅活細胞週期檢測點激酶1(Chk1)和細胞週期檢測點激酶2(Chk2)基因對DDP誘導的肺癌細胞凋亡的影響.方法 優化轉染條件後,將肺癌A549細胞分為8組進行轉染,分彆為正常組(未經任何處理的A549細胞)、對照組(A549細胞接受10 μmol/L的DDP作用12 h)、轉染Chk1正義寡覈苷痠鏈(sODN)組、轉染Chk1反義寡覈苷痠鏈(AsODN)組、轉染Chk2 sODN組、轉染Chk2 AsODN組、聯閤轉染Chk1和Chk2 sODN組以及聯閤轉染Chk1和Chk2 AsODN組,其中後6組轉染24 h後再以10 μmol/L的DDP處理12 h.採用逆轉錄聚閤酶鏈反應(RT-PCR)和Western blot法,檢測轉染Chk1或Chk2 sODN、AsODN後,A549細胞中Chk1或Chk2 mRNA和蛋白的錶達.採用流式細胞儀Annexin V-FITC法和Sub-G1法,檢測轉染Chk1和(或)Chk2 sODN、AsODN後,DDP作用下A549細胞的細胞週期和凋亡變化.結果 10μmol/L的DDP作用12 h後,非同步化處理的A549細胞齣現瞭明顯的S期阻滯現象.轉染24 h後,Chk1或Chk2 AsODN可明顯抑製A549細胞中Chk1 mRNA和蛋白或Chk2 mRNA和蛋白的錶達.與單獨轉染Chk1或Chk2 sODN組相比,單獨轉染Chk1或Chk2 AsODN組DDP誘導下A549細胞的凋亡率約提高瞭1～2倍(P＜0.05);但聯閤轉染Chk1和Chk2 AsODN組與單獨轉染Chk1 AsODN或Chk2 AsODN組相比,A549細胞凋亡率的差異無統計學意義(P=0.521和P=0.339).結論 滅活Chk1和Chk2基因引起化療增敏的機製可能是通過解除S期阻滯這種腫瘤細胞的自我保護機製實現的,Chk1和Chk2基因可作為肺癌化療藥物增敏治療的有效靶點.
목적 관찰순박(DDP)작용하폐암A549세포계적세포주기화조망적동역학특점,이급멸활세포주기검측점격매1(Chk1)화세포주기검측점격매2(Chk2)기인대DDP유도적폐암세포조망적영향.방법 우화전염조건후,장폐암A549세포분위8조진행전염,분별위정상조(미경임하처리적A549세포)、대조조(A549세포접수10 μmol/L적DDP작용12 h)、전염Chk1정의과핵감산련(sODN)조、전염Chk1반의과핵감산련(AsODN)조、전염Chk2 sODN조、전염Chk2 AsODN조、연합전염Chk1화Chk2 sODN조이급연합전염Chk1화Chk2 AsODN조,기중후6조전염24 h후재이10 μmol/L적DDP처리12 h.채용역전록취합매련반응(RT-PCR)화Western blot법,검측전염Chk1혹Chk2 sODN、AsODN후,A549세포중Chk1혹Chk2 mRNA화단백적표체.채용류식세포의Annexin V-FITC법화Sub-G1법,검측전염Chk1화(혹)Chk2 sODN、AsODN후,DDP작용하A549세포적세포주기화조망변화.결과 10μmol/L적DDP작용12 h후,비동보화처리적A549세포출현료명현적S기조체현상.전염24 h후,Chk1혹Chk2 AsODN가명현억제A549세포중Chk1 mRNA화단백혹Chk2 mRNA화단백적표체.여단독전염Chk1혹Chk2 sODN조상비,단독전염Chk1혹Chk2 AsODN조DDP유도하A549세포적조망솔약제고료1～2배(P＜0.05);단연합전염Chk1화Chk2 AsODN조여단독전염Chk1 AsODN혹Chk2 AsODN조상비,A549세포조망솔적차이무통계학의의(P=0.521화P=0.339).결론 멸활Chk1화Chk2기인인기화료증민적궤제가능시통과해제S기조체저충종류세포적자아보호궤제실현적,Chk1화Chk2기인가작위폐암화료약물증민치료적유효파점.
Objective 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.
