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目的观察人PIF1解旋酶敲低对电离辐射致细胞生长及细胞周期阻滞的影响,以探讨人PIF1在电离辐射致DNA损伤应答中的作用。方法采用慢病毒系统建立PIF1敲低稳转细胞系,实时荧光定量PCR及Western印迹检测PIF1敲低效果。细胞计数法观察PIF1敲低对4 Gyγ射线照射下细胞增殖的影响,流式细胞术观察PIF1敲低对8 Gyγ射线照射下细胞周期阻滞的影响。结果建立了PIF1敲低的稳转细胞系,4 Gyγ射线照射后,PIF1敲低细胞系的生长受到明显抑制,在照后第2~4天几乎无任何增殖,直到第5天才开始缓慢增殖。8 Gyγ射线照射后,PIF1敲低细胞系的S期阻滞与G_2/M期阻滞相对于对照细胞发生明显延迟。结论 PIF1敲低后显著增加了细胞的辐射敏感性以及电离辐射所致的S期阻滞与G_2/M期阻滞延迟。
Objective To investigate the effect of human PIF1 knockdown on cell growth and cell cycle arrest induced by ionizing radiation in order to investigate the role of human PIF1 in response to DNA damage induced by ionizing radiation. Methods The lentiviral system was used to establish the PIF1 knockdown stable cell line. The knockdown effect of PIF1 was detected by real - time fluorescence quantitative PCR and Western blotting. Cell counting was used to observe the effect of PIF1 knockdown on cell proliferation under 4 Gy γ-ray irradiation. The effect of PIF1 knockdown on cell cycle arrest under 8 Gy γ-ray irradiation was observed by flow cytometry. Results PIF1 knockdown stable cell lines were established. After 4 Gy γ-ray irradiation, the growth of PIF1 knockdown cell line was significantly inhibited. There was almost no proliferation in the 2nd to 4th days after irradiation, and began to proliferate slowly on the 5th day. After 8 Gy gamma irradiation, S phase arrest and G 2 / M phase arrest of PIF1 knockdown cell line were significantly delayed compared with that of control cells. Conclusion The knockdown of PIF1 significantly increased the radiosensitivity of cells and the delay of S phase arrest and G 2 / M arrest induced by ionizing radiation.