P53在电离辐射诱导的细胞周期解耦联中的作用

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,4 Gy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。

Roles of p53 in ionizing radiation-induced cell cycle uncoupling

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^{- -} cells. Compared with p53^{+ +}+IR group, the decrease of G₀ G₁ 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.