p27^(Kip1)在As_2O_3诱导白血病细胞凋亡中的作用机制

The mechanism of p27^(Kip1) in leukemia cell apoptosis induced by As_2O_3

目的:研究三氧化二砷(arsenic trioxide,As_2O_3)及转录生长因子(transforming growth factor-β1,TGF-β1)对人HL-60细胞增殖的影响,并探讨相关信号通路。方法:流式细胞术检测细胞周期及凋亡,RTPCR检测p27^(Kip1)、TGF-β1、Cyclin E和Bcl-2表达,免疫组织化学方法和Western blot方法检测p27^(Kip1)、TGF-β1、Cyclin E和Bcl-2蛋白水平。结果:As_2O_3、TGF-β1单药或联合处理均可诱导细胞凋亡,以联合处理组凋亡最明显,并且As_2O_3单药组及联合处理组细胞周期阻滞于G1期。As_2O_3单药及联合处理组可见p27^(Kip1)、内源性TGF-β1表达上调,Cyclin E和Bcl-2表达下调,外源性TGF-β1处理组可见p27^(Kip1)mRNA及蛋白表达上调,内源性TGF-β1 mRNA表达上调,内源性TGF-β1蛋白水平与对照组比较无明显变化,Cyclin E和Bcl-2表达下调,联合处理组p27^(Kip1)及内源性TGF-β1表达上调及Cyclin E和Bcl-2表达下调程度强于单药处理组。结论:As_2O_3诱导细胞凋亡的机制可能是通过上调TGF-β1,从而上调p27^(Kip1),拮抗Cyclin E和Bcl-2的作用,抑制细胞增殖,使细胞周期阻滞于G_1期,从而诱导细胞发生凋亡,外源性TGF-β1通过上调内源性TGF-β1,从而上调p27^(Kip1),增强As_2O_3诱导细胞凋亡的作用。

Objective：To study the proliferation and apoptosis of arsenic trioxide （arsenic trioxide, As2 03 ） and （transforming growth factor beta 1, TGF- β1 ） on HL- 60 cell, as well as discuss the related mechanism. Methods： HL -60 cells treared with different concentrations As2 03 , trypan blue staining detect cell proliferation inhibition rate, inhibition rate of HL- 60 treated with AszO3 and （or） TGF- β1 was deteated with CCK- 8, and cell cycle was de- tected with flow cytometry, p27Kipl , endogenous TGF - β1, Cyclin E and Bcl -2 were detected by SYBR GreenRT - PCR,immunohistochemieal method and Western blotting. Results： Apoptosis of HL - 60 cells induced by different concentrations of As203 produced a time -dose dependent relationship. As203 and （or） TGF - β1 can induce apopto- sis on HL -60 cells, and the combination group had the most obvious apoptosis. Cell cycle was arrested at Gl phase in As2 03 single treatment group and combined treatment group, p27KIpl and endogenous TGF - β1 of HL -60 cells trea- ted by As2 03 single and combined with exogenous TGF - β1 were increased, accompaning with decreased expression of Cyclin E and Bcl -2, p27Kip1 was upregulated after treated exogenous TGF - β1, and the mRNA expression of en- dogenous TGF- β1 was upregulated, but the protein expression of endogenous TGF - β1 has no significant change compared with the control group, Cyelin E and Bcl- 2 were downregulated in the exogenous TGF- β1 group, the ex- pression change was stronger in combined group than in the single drug treatment. Conelusion：Apoptosis of HL -60 cells was induced by AszO3 and （or） TGF -β1 ,and the cell cycle was arrested at Gt phase in As203 single treatment group and combined treatment group. Apoptosis of HL-60 cells induced by As203 may be caused by up- regulating TGF - β1 ,then 1027Kipl ,which was antagonistic to Cyclin E and Bcl -2 ,leading to the cell cycle arrested at G1 phase. Exogenous TGF - β1 can enhance the role of ms203 by upregulate the endogenous TGF - β1.