P53在电离辐射诱导的细胞周期解耦联中的作用

Roles of p53 in ionizing radiation-induced cell cycle uncoupling

目的探讨p53基因在电离辐射（IR）诱导的MCF-7细胞周期解耦联中的作用。方法构建RNAi表达载体，经磷酸钙共沉淀法转染293T细胞形成病毒包装颗粒，感染MCF-7后采用Western blot检测P53蛋白的表达，建立p53基因沉默模型。将p53野生型（＋／＋）和沉默模型（-／-）经电离辐射处理后，采用流式细胞术分别测定细胞周期并分析细胞多倍体的变化。结果与p53^＋/＋组比较，p53^-/-模型组G0/G1期细胞百分数减少，S期、G2期增加（P〈0．01），倍体分析表明二倍体数减少，四倍体、八倍体均增加（P〈0．01）。在p53^＋/＋和p53^-/-细胞中，与假照射组比较，4Gy照射后G0／G1期、S期细胞百分数减少，而G2期增多（P〈0．01）；倍体分析表明，照射后二倍体数减少，四倍体、八倍体均增加（P〈0．01）。与p53^＋/＋IR组比较，p53^-/-＋IR组发生G0／G1期、S期细胞百分数减少，G2＋M期增多（P〈0．01），二倍体数减少，四倍体增多（P〈0．01），八倍体无明显差别。结论电离辐射可以诱导细胞发生G2期阻滞和细胞周期解耦联；P53在电离辐射诱导的MCF-7细胞G2期阻滞中发挥作用，而在细胞周期解耦联中可能不发挥作用。

Objective To explore the roles of p53 in ionizing radiation induced MCF-7 cell cycle uncoupling. Methods The p53 knock-down models was established in MCF-7 with retrovirus packaged particles from 293T cells through calcium acid phosphate co-precipitation, then Western blot was used to detect the protein expression. Flow cytometry（FCM） was used to analyze the cell cycle uncoupling and polyploid after irradiation. Results Compared with p53^＋/＋ group, the percentages of G0/G1 cells in p53^-/- group decreased, while those of S and G2 ＋ M increased （ P 〈 0.01 ） . In polyploidy analysis 2N cells decreased, whereas both 4N and 8N cells increased （ P 〈 0.01）. Compared with sham-irradiation, 4 Gy X-ray led to the decrease of G0/G1 , S cells, and the increase of G2 ＋ M cells. The increase of 2N cells and decrease of 4N and 8N cells were observed in both p53^＋/＋ and p53^-/- ceils. Compared with p53＋/＋ ＋ IR group, the decrease of G0/C1 and S cells and the increase of G2 ＋ M cells were significant （ P 〈 0.01 ） in p53^-/- ＋ IR groups. 2N cells decreased, 4N cells increased, but no changes in 8 N cells occurred. Conclusion Radiation might induce G2 arrest and cycle uncoupling, p53 plays a role in the regulation of G2 arrest, but no role in cycle uncoupling.