摘要
目的:研究重组腺病毒介导的野生型p53基因(Ad5wtp53)对人慢性粒细胞白血病K562细胞增殖的影响及其可能机制.方法:分别将含有Ad5wtp53,突变型p53基因(Ad5mtp53)和绿色荧光蛋白基因的重组腺病毒表达载体(Ad5GFP),联合阳离子多聚季胺(polybrene)共感染K562细胞,经RT-PCR和Western Blot检测p53mRNA和P53蛋白的表达后,应用流式细胞术分析K562细胞周期、DNA含量的变化.WesternBlot检测P21蛋白、核磷蛋白(NPM)、磷酸化核磷蛋白(Thr199-NPM)的表达情况.结果:Ad5wtp53和Ad5mtp53能在K562细胞中表达p53mRNA和P53蛋白.Ad5wtp53感染K562细胞48,72h后分别有(74.45±12.09)%和(69.80±11.70)%的细胞周期出现G0/G1期阻滞,DNA含量分别下降了32.25%和38.13%;同时P21蛋白的表达升高,核磷蛋白的表达无改变,磷酸化核磷蛋白(Thr199)表达下降.而Ad5mtp53感染组和Ad5GFP感染组未见相应改变.结论:野生型P53蛋白可能通过上调P21的表达,阻滞细胞周期于G0/G1期,抑制S期DNA的合成,降低核磷蛋白第199位苏氨酸残基的磷酸化来抑制K562细胞的增殖.
AIM： To observe the effect of recombinant adenovirus-mediated wild-type p53 gene （ Ad5wtp53 ） on proliferation of leukemia K562 cells and to explore its possible mechanism in chronic myeloid leukemia in blastic crisis. METHODS： The recombinant adenoviruses carrying Ad5wtp53, mutational p53 gene （Ad5mtp53） and the green fluorescent protein （Ad5GFP） gene were co-infected into K562 cells with cation polybrene. After the expressions of p53 gene were determined by RT-PCR and Western Blot respectively, the cell cycle and DNA content of K562 cells were both analyzed by flow cytometry. The protein expressions of P21, nucleophosmin （ NPM ） and phospho-NPM （Tbr199） were determined by Western Blot respectively. RESULTS： There were sustained expressions of p53 mRNA and protein in K562 cells infected by Ad5wtp53 and Ad5mtp53. There were （74.45 ± 12.09）% and （69.80 ± 11.70）% arrest of the cell cycle at the G0/G1 phase,and 32.25% and 38.13% decline of DNA content observed after 48 and 72 h, and increased P21 protein expression and decreased pbospho-NPM （Thr199） protein expression were also observed ,while NPM protein didn＇t change after the infection of K562 ceils by Ad5wtp53; the groups of Ad5mtp53 and Ad5GFP had no changes. CONCLUSION： Wildtype P53 protein may contribute to the up-regulation of P21 protein expression, the arrest of cell cycle in G0/G1 phase, the inhibition of DNA synthesis at S phase, as well as the down-regulation of Thr199-NPM phosphorylation; all of these inhibit the excessive proliferation of K562 cells.
出处
《第四军医大学学报》
北大核心
2008年第21期1955-1958,共4页
Journal of the Fourth Military Medical University
关键词
慢性粒细胞白血病
P53基因
中心体
leukemia, myelocytics, chronic
eentrosome
p53 gene
K562 cells
NPM
phosphorylation
