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目的:乳腺癌是严重威胁妇女健康的重要疾病,放疗常常作为乳腺癌综合治疗的重要手段。研究表明,临床上产生放疗抵抗的主要原因是由于放疗激活细胞周期检测点信号传导通路引起细胞自我修复而逃避凋亡,因而目前通过抑制细胞周期检测点信号通路增强肿瘤放、化疗敏感性,已成为国际上抗肿瘤治疗的一个重要方向和研究热点。Chk1和Chk2是细胞周期检测点中最重要的丝氨酸/苏氨酸激酶,本研究通过反义封闭Chk1和/或Chk2基因,阻断细胞周期检测点信号传导通路,研究对乳腺癌MDA-MB-231细胞放疗后细胞周期和凋亡的影响,从而评价Chk1和Chk2基因作为肿瘤治疗靶点的有效性。方法:Western-Blot法检测转染Chk1和Chk2正、反义寡核苷酸后,细胞内Chk1和Chk2蛋白表达情况;流式细胞仪AnnexinV-PI法和SubG1法检测单转染或联合转染Chk1/2反义寡核苷酸对放疗后MDA-MB-231细胞凋亡和细胞周期的影响。结果:反义封闭Chk1或Chk2基因可解除G2/M期阻滞,增强放疗后凋亡敏感性,而同时反义封闭Chk1和Chk2基因具有协同作用。结论:阻断细胞周期检测点信号通路的关键激酶Chk1和Chk2可显著增强放疗后凋亡敏感性。
OBJECTIVE: Breast cancer is an important disease that seriously threatens the health of women. Radiotherapy is often used as an important means of comprehensive treatment of breast cancer. Studies have shown that the main reason for clinical radiation resistance is due to radiation-activated cell cycle checkpoint signaling pathway to cause cells to self-repair and escape from apoptosis, and thus inhibit the cell cycle checkpoint signaling pathway to enhance tumor radiotherapy and chemotherapy sensitivity has been Become an international anti-cancer treatment is an important direction and research hot spots. Chk1 and Chk2 are the most important serine / threonine kinases in the cell cycle checkpoint. In this study, we blocked the signal transduction pathway of the cell cycle checkpoint by blocking the Chk1 and / or Chk2 gene antisense, 231 cells after radiotherapy cell cycle and apoptosis, in order to evaluate the Chk1 and Chk2 gene as a tumor therapeutic target effectiveness. Methods: The expressions of Chk1 and Chk2 in Chk1 and Chk2 were detected by Western-Blot. The expression of Chk1 and Chk2 were detected by Western Blot and AnnexinV-PI by flow cytometry and SubG1 method. Effect of Chk1 / 2 antisense oligonucleotide on apoptosis and cell cycle of MDA-MB-231 cells after radiotherapy. RESULTS: Antisense blocking of Chk1 or Chk2 gene could relieve the G2 / M arrest and enhance the apoptosis sensitivity after radiotherapy. At the same time, antisense blocking Chk1 and Chk2 genes had synergistic effect. Conclusion: Chk1 and Chk2, which can block the signal transduction pathway of cell cycle checkpoint, can significantly enhance the apoptosis sensitivity after radiotherapy.