野生型p53对白血病K562细胞中心体过度复制的抑制作用

背景与目的：肿瘤组织细胞中p53基因突变或缺失是导致非整倍体的发生和基因组不稳定的主要原因之一：最近研究发现慢性粒细胞白血病（chronic myelogenous leukemia．CML）各期患者均有中心体异常，且异常的程度与临床分期有关。急变期显示更为严重的中心体异常。本研究建立携野生型p53基因的CML急变K562细胞株．以研究该细胞内野生型p53基因表达后p53信号转导通路对K562细胞中心体的影响。方法：用HEK293细胞扩增重组p53野生型、突变型及空载腺病毒载体，联合polybrene分别感染K562细胞；未接受感染的细胞作为空白对照。流式细胞术检测重组腺病毒载体感染效率，Western blot检测P53蛋白表达。间接免疫荧光染色后用激光共聚焦计数K562细胞中心体的变化。Westernblot检测p53信号转导通路下游效应分子生长阻滞和DNA损伤应答基因Gadd45a（growtharrest and DNA damage）、BubRl（Bublrelated）、AuroraA的表达。结果：成功建立携野生型p53基因的K562细胞株．腺病毒载体感染效率达60％以上，野生型p53可在K562细胞中持续表达。感染72h后，携野生型p53基因的K562细胞中中心体数量异常（n〉2）的细胞比例降至（0．38＋0．02）％，与空白对照组（0．71＋0．14）％比较其差异有统计学意义（P〈0．05）；Westernblot结果发现p53信号转导通路下游效应分子Gadd45a、BubRl表达分别上调93％、88％．而AuroraA的表达下降56％（P值均〈0．05）。结论：重组腺病毒介导的野生型p53基因能够在白血病K562细胞中持续表达：野生型P53蛋白可能通过转录激活-依赖途径上调Gadd45a、BubR1表达以及转录激活-非依赖途径使AuroraA的表达下降，从而抑制K562细胞中心体的过度复制。

Inhibitory effect of wild-type p53 gene on excessive replication of centrosomes in leukemia cell line K562

Background and Objective： Mutation and deletion of p53 gene in tumor cells is one of the major reasons for aneuploid development and genomic instability. Abnormal centrosomes exist in chronic myelogenous leukemia patients at different stages; furthermore, the degree of abnormality is associated with clinical stage and more severe in blast crisis stage. This study was to establish the leukemia cell line K562 with exogenous wild-type p53（wt-p53） gene, and explore the effect of p53 gene on centrosomes in K562 cells. Methods. The recombinant adenoviruses carrying wt-p53 gene （Ad5wtp53）, mutant p53 gene （Ad5mtp53） and green fluorescent protein gene （Ad5GFP） were amplified respectively in HEK293 cells, and co-infected with cation polybrene into K562 cells respectively; uninfected K562 cells were used as blank control. The infection efficiency was analyzed by flow cytometry. P53 expression was detected by Western blot. Centrosomes were counted under the laser confocal microscope after indirect immunofluorescence staining. The expression of Gadd45a （growth arrest and DNA damage）, BubR1 （Bub 1 related）, and Aurora A was detected by Western blot. Results： K562 cell line with exogenous wt-p53 gene was established. The infection efficiencies of three groups were over 60%, and P53 sustained to express for 72 h. The percentage of cells with amplified centrosomes （more than 2/cell） in Ad5wtp53 group was decreased to （0.38±0.02）%, lower than that of blank control group （P〈0.05）. Meanwhile, the protein levels of Gadd45a and BubR1 in Ad5wtp53 group were up-regulated by 93% and 88% of blank control （P〈0.05） respectively, and the protein level of Aurora A was down-regulated by 56% of blank control （P〈0.05）. Conclusions： P53 protein is sustained to express in K562 cells after being infected by Ad5wtp53. wt-p53 can suppress excessive replication of centrosomes that might contribute to the up-regulation of Gadd45a and BubR1 protein expression as well as the down-regulation of Aur