中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2009年
3期
178-182
,共5页
高庆蕾%叶飞%邢辉%谢大兴%卢运萍%周剑锋%马丁
高慶蕾%葉飛%邢輝%謝大興%盧運萍%週劍鋒%馬丁
고경뢰%협비%형휘%사대흥%로운평%주검봉%마정
辐射耐受性%Chk1基因%Chk2基因%HeLa细胞系%细胞周期%凋亡
輻射耐受性%Chk1基因%Chk2基因%HeLa細胞繫%細胞週期%凋亡
복사내수성%Chk1기인%Chk2기인%HeLa세포계%세포주기%조망
Radiation tolerance%Chk1 gene%Chk2 gene%HeLa cell line%Cell cycle%Apoptosis
目的 探讨下调Chk1和Chk2基因对HeLa细胞射线照射后细胞周期和凋亡的影响及其作用机制.方法 应用流式细胞仪检测不同剂量60Co照射引起的HeLa细胞周期和凋亡的动力学变化,以激光共聚焦显微镜和Western blot法检测转染Chk1和Chk2正义寡核苷酸(sODN)和反义寡核苷酸(AsODN)对Chk1和Chk2蛋白表达的影响,以AnnexinV-PI法、凋亡细胞的周期特异性检测法及透射电镜观察单独或联合转染Chk1和Chk2反义寡核苷酸对射线照射后HeLa细胞凋亡的影响.结果 不同剂量的60Co照射可引起Hela细胞小同程度的G2/M期阻滞,15 Gy的60Co照射48 h后,G2/M期细胞可达75.53%±3.72%.当细胞周期阻滞解除后,细胞凋亡明显增加.转染Chk1 AsODN组的早期凋亡、晚期凋亡和死亡细胞共有94.42%±4.78%,明显高于转染Chk1 sODN组(44.35%±2.08%,P<0.0001);转染Chk2 AsODN组的凋亡细胞共有93.08%±5.24%,明显高于转染Chk2 sODN组(48.98%±3.35%,P<0.0001);而共转染Chk1和Chk2 AsODN组的凋亡细胞共有94.26%±4.92%,明显高于共转染Chk1和Chk2 sODN组(42.46%±2.56%,P<0.0001);共转染Chk1和Chk2AsODN组与仅转染Chk1 AsODN或Chk2 AsODN组比较,差异无统计学意义(P>0.05).凋亡细胞的周期特异性检测结果显示,转染Chk1和Chk2 sODN引起的凋亡主要来自于G1期细胞,而转染Chk1 AsODN或Chk2 AsODN后,G1期、S期、G2/M期的凋亡细胞均较对应sODN转染组明显增加,尤其共转染Chk1和Chk2 AsODN组更为显著.结论 射线照射激活细胞周期检测点信号传导通路引起细胞自我保护,是临床上产生放疗抵抗的主要原因,而阻断该信号通路的关键激酶Chk1或Chk2,可显著增强细胞的放射敏感性,其机制在于改变了凋亡细胞的周期特异性,凋亡细胞可以来自G1、S、G2/M各周期的细胞.
目的 探討下調Chk1和Chk2基因對HeLa細胞射線照射後細胞週期和凋亡的影響及其作用機製.方法 應用流式細胞儀檢測不同劑量60Co照射引起的HeLa細胞週期和凋亡的動力學變化,以激光共聚焦顯微鏡和Western blot法檢測轉染Chk1和Chk2正義寡覈苷痠(sODN)和反義寡覈苷痠(AsODN)對Chk1和Chk2蛋白錶達的影響,以AnnexinV-PI法、凋亡細胞的週期特異性檢測法及透射電鏡觀察單獨或聯閤轉染Chk1和Chk2反義寡覈苷痠對射線照射後HeLa細胞凋亡的影響.結果 不同劑量的60Co照射可引起Hela細胞小同程度的G2/M期阻滯,15 Gy的60Co照射48 h後,G2/M期細胞可達75.53%±3.72%.噹細胞週期阻滯解除後,細胞凋亡明顯增加.轉染Chk1 AsODN組的早期凋亡、晚期凋亡和死亡細胞共有94.42%±4.78%,明顯高于轉染Chk1 sODN組(44.35%±2.08%,P<0.0001);轉染Chk2 AsODN組的凋亡細胞共有93.08%±5.24%,明顯高于轉染Chk2 sODN組(48.98%±3.35%,P<0.0001);而共轉染Chk1和Chk2 AsODN組的凋亡細胞共有94.26%±4.92%,明顯高于共轉染Chk1和Chk2 sODN組(42.46%±2.56%,P<0.0001);共轉染Chk1和Chk2AsODN組與僅轉染Chk1 AsODN或Chk2 AsODN組比較,差異無統計學意義(P>0.05).凋亡細胞的週期特異性檢測結果顯示,轉染Chk1和Chk2 sODN引起的凋亡主要來自于G1期細胞,而轉染Chk1 AsODN或Chk2 AsODN後,G1期、S期、G2/M期的凋亡細胞均較對應sODN轉染組明顯增加,尤其共轉染Chk1和Chk2 AsODN組更為顯著.結論 射線照射激活細胞週期檢測點信號傳導通路引起細胞自我保護,是臨床上產生放療牴抗的主要原因,而阻斷該信號通路的關鍵激酶Chk1或Chk2,可顯著增彊細胞的放射敏感性,其機製在于改變瞭凋亡細胞的週期特異性,凋亡細胞可以來自G1、S、G2/M各週期的細胞.
목적 탐토하조Chk1화Chk2기인대HeLa세포사선조사후세포주기화조망적영향급기작용궤제.방법 응용류식세포의검측불동제량60Co조사인기적HeLa세포주기화조망적동역학변화,이격광공취초현미경화Western blot법검측전염Chk1화Chk2정의과핵감산(sODN)화반의과핵감산(AsODN)대Chk1화Chk2단백표체적영향,이AnnexinV-PI법、조망세포적주기특이성검측법급투사전경관찰단독혹연합전염Chk1화Chk2반의과핵감산대사선조사후HeLa세포조망적영향.결과 불동제량적60Co조사가인기Hela세포소동정도적G2/M기조체,15 Gy적60Co조사48 h후,G2/M기세포가체75.53%±3.72%.당세포주기조체해제후,세포조망명현증가.전염Chk1 AsODN조적조기조망、만기조망화사망세포공유94.42%±4.78%,명현고우전염Chk1 sODN조(44.35%±2.08%,P<0.0001);전염Chk2 AsODN조적조망세포공유93.08%±5.24%,명현고우전염Chk2 sODN조(48.98%±3.35%,P<0.0001);이공전염Chk1화Chk2 AsODN조적조망세포공유94.26%±4.92%,명현고우공전염Chk1화Chk2 sODN조(42.46%±2.56%,P<0.0001);공전염Chk1화Chk2AsODN조여부전염Chk1 AsODN혹Chk2 AsODN조비교,차이무통계학의의(P>0.05).조망세포적주기특이성검측결과현시,전염Chk1화Chk2 sODN인기적조망주요래자우G1기세포,이전염Chk1 AsODN혹Chk2 AsODN후,G1기、S기、G2/M기적조망세포균교대응sODN전염조명현증가,우기공전염Chk1화Chk2 AsODN조경위현저.결론 사선조사격활세포주기검측점신호전도통로인기세포자아보호,시림상상산생방료저항적주요원인,이조단해신호통로적관건격매Chk1혹Chk2,가현저증강세포적방사민감성,기궤제재우개변료조망세포적주기특이성,조망세포가이래자G1、S、G2/M각주기적세포.
Objective To explore the increasing effect of blocking Chk1 and/or Chk2 gene by Chk1 or Chk2-specific antisense oligodeoxynucleotides (AsODN) on apoptosis in HeLa cell line after irradiation and its mechanism of action. Methods Asynchronized HeLa cells were exposed to 60Co-irradiation at different dosage to activate G2/M checkpoint arrest. The cell cycle profiles were observed in HeLa cells after irradiation at a range of various doses and different time points by flow cytometry. In the experimental groups, Chk1/2 sODN and AsODN alone or in combination were transfected into HeLa cells, and the cells were exposed to 60Co-irradiation at 24 h after transfection. The changes of Chk1/2 protein expression were assayed by Western blot and confocal laser scanning microscopy (Confocal), and the cell cycles, apoptosis rates and cell cycle specific apoptosis were detected by annexin V-PI labeling and flow cytometry. Results Apoptotic response was significantly increased in the Hela cells after G60/M arrest and was inversed to activation of G2/M checkpoint. Either Chk1 or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptosis by 90%～120%, compared to corresponding sODN control (P<0.05). Unexpectedly, combined use of Chk1-and Chk2-spocific AsODN did not produce synergistic effect as compared to treatment with Chk1-or Chk2-specific AsODN alone (P>0.05). While irradiated HeLa cells underwent apoptosis preferentially in G1-phase, apoptosis occurred in either of G1-, S-or G2/M-phase in the presence of Chk1 and/or Chk2 AsODN. Conclusion The radioresistance is mainly induced by activating the cell cycle checkpoint signal transduction pathway after irradiation, and abrogating of the key effector Chk1 and Chk2 may increase the apoptotic sensitivity to irradiation due to changes of the pattern of cell cycle specific apoptosis.
