下调Chk1和Chk2基因促进射线照射后HeLa细胞凋亡

Down-regulation of Chk1/Chk2 gene expression increases apoptosis in irradiated HeLa cells and its mechanism

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细胞不同程度的G2／M期阻滞，15 Gy的^60Co照射48h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chk2 AsODN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。

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 G2/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-specific 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.