中文摘要：

目的探索结肠癌细胞株SW480在长期分割放疗下获得放射抗拒性的可能机制。方法经不同剂量0Gy、2Gy、5Gy、10Gy单次x射线照射后，以RT．PCR法检测结肠癌亲本细胞SW480和放射抗拒性细胞SW480-R中CCNDlmRNA的表达；Westernblot法检测DNA．PK／AKT／GSK3[3通路中两种细胞株cyclinD1、CDK4、Rb、P—Rb．Ser795、AKT、p-AKT-ser473、GSK313、p-GSK3β-Ser9、DNA．PKcs、P—DNA．PKcs蛋白的表达。结果经单次x射线照射0Gy、2Gy、5Gy、10Gv后，RT—PCR法检测显示SW480．R细胞中CCNDlmRNA的表达明显低于SW480的表达（P〈0．05），其相对表达水平分别为0．31±0．02、0．32±0．03、0．34＋0．05、0．44±0．04；Westernblot法检测显示SW480和SW480．R中的cyclinDI、CDK4、Rb、P—Rb．Ser795、AKT、P—AKT．Ser473、p-GSK3β-Ser9、DNA—PKcs及p-DNA-PKcs蛋白的表达含量在两种细胞中显著不同，SW480．R的蛋白表达明显高于SW480（P〈0．05），而GSK313蛋白的表达含量SW480．R却低于SW480（P〈0．05）。结论经过长期分割放疗的结肠癌细胞获得的放射抗拒性可能与激活DNA—PK／AKT／GSK313通路中cyclinD1蛋白过表达介导的DNA损伤反应（DDR）的改变有关。

英文摘要：

Objective To investigate the molecular mechanism of radioresistance acquisition after long-term fractionated radiation （FR） in human colon cancer cell llne SW480. Methods Parental SW480 cells and radioresistant subline SW480-R cells were irradiated with single doses of 0,2,5 or 10 Gy.CCND1 mRNA levels were measured using real-time fluorescent quantitative polymerase chain reaction（PCR）.Western blotting was used to measure levels of proteins involved in the DNA-PK/AKT/GSK3β pathway of SW480 cells and SW480-R cells,including cyclin D1, CDK4, Rb,p-Rb-Sebr795, AKT, p-AKT-Ser473, GSK3β, p-GSK3β-Serg, DNA-PKcs and p-DNA-PKcs. Results CCND1 mRNA levels were significantly lower in the radioresistant subline SW480-R than in parental SW480 cells after single irradiation doses （P〈0.05）,with relative expression being 0.31±0.02 in the resistant subline compared to the parental line after 0 Gy,0.32±0.03 after 2 Gy,0.34±0.05 after 5 Gy and 0.44±0.04 after 10 Gy.Conversely levels of all proteins examined were higher in SW480-R cells than in SW480 cells（P〈0.05） except the level of GSK3β, which was lower in SW480-R cells（P〈0.05）. Conclusions Acquired radioresistance in the human colon cancer cell line was correlated with alterations in the DNA damage response;these alterations were activated by long-term FR and mediated by cyclin D1 overexpression in the DNA-PK/AKT/GSK3β pathway.