P27^(Kip1)和TGF-β1在原代APL细胞凋亡中作用机制的研究

Role of P27^(Kip1) and TGF-β1 in APL Cell Apoptosis Induced by As_2O_3

本研究探讨三氧化二砷(arsenictrioxide,As2O3)对原代APL细胞凋亡及周期的影响,As2O2作用前后P27Kip1、TGF-β1、cyclinE和bcl-2的变化以及P27Kip1在As2O3诱导急性早幼粒细胞白血病细胞凋亡中的作用。用细胞形态学及流式细胞仪分别检测原代APL细胞凋亡和细胞周期改变,免疫组织化学法和RT-PCR技术检测药物处理前后细胞P27Kip1、TGF-β1、cyclinE以及BCL-2蛋白和基因表达的变化。结果表明:As2O3与APL细胞共同培养24小时,浓度为1、5、10μmol/L时,凋亡细胞所占比例分别为1.42%、4.57%、10.67%;至48小时,凋亡细胞分别为8.92%、16.07%和18.90%,明显高于相同浓度的As2O3作用24小时(p<0.05);至72小时,细胞以碎片为主。同一时段随着As2O3浓度的增加,APL细胞凋亡不断增多;同一浓度As2O3作用于APL细胞,随着作用时间的延长,APL细胞凋亡也随之增多。5、10μmol/L的As2O3作用于APL细胞24、48、72小时可见细胞周期阻滞于G1期,G1期细胞比例均明显高于对照组(p<0.05),而1μmol/LAs2O3作用后的细胞周期无明显变化。1、5和10μmol/LAs2O3作用于APL细胞,P27Kip1蛋白表达增高,P27Kip1mRNA与β-actin灰度值之比(P27Kip1/β-actin)分别为0.181、0.489和0.921,表明随着As2O3浓度的增高,P27Kip1mRNA表达水平显著上调(p<0.05),P27Kip1表达与凋亡细胞数之间存在正相关(r=0.55,p<0.05);与对照组相比,TGF-β1蛋白和mRNA表达上调,伴有CyclinE、BCL-2蛋白和mRNA表达下调,TGF-β1表达与凋亡细胞数之间存在正相关(r=0.51,p<0.05),TGF-β1表达与P27Kip1表达之间也存在正相关(r=0.31,p<0.05)。结论:As2O3体外可诱导原代APL细胞凋亡,存在时间-剂量依赖关系,并使细胞周期阻滞于G1期。P27Kip1表达的变化情况与As2O3诱导细胞凋亡的作用效果密切相关,As2O3诱导原代APL细胞凋亡的机制可能是通过上调TGF-β1,从而上调P27Kip1,拮抗CyclinE和BCL-2的作用,抑制细胞增殖,使细胞周期阻滞于G1期,从而诱导细胞发生凋亡。

The aim of study was to investigate the effects of arsenic trioxide （ As2O3 ） on cell cycle and apoptosis of APL cells, as well as changes of P27Kip1 , endogenous TGF-β1, cyclin E and bcl-2, and to explore the relationship between expression of P27Kip1 and apoptosis induced by As2O3. The apoptosis and cell cycle changes of APL cells treated with As2O3 were detected by morphology and flow cytometry respectively, the protein and mRNA expressions of P27^Kip1 ,TGF-β1 ,cyclin E and BCL-2 were measured by immunohistochemistry and RT-PCR. The results indicated that As2O3 induced APL cell apoptosis in vitro, and cell cycle was arrested at G1 phase. Apoptotic cells induced by As2O3 1, 5 and 10 μmol/L for 24 hours were 1.42%, 4.57% and 10.67% respectively; the proportion of apoptotic cells induced by As2O3 of same concentrations for 48 hours increased m 8.92%, 16.07% and 18.90% respectively ; the cells induced by As203 for 72 hours were mainly in debris. Protein and mRNA expressions of P27Kip1 and TGF-β1 of APL cells after treatment with As2O3 increased, accompaning with decrease of cyclin E, bcl-2 protein and mRNA expressions. Apoptot- ic cells were related to the expressions of P27Kip1 （ rmRNA = 0. 55, p 〈 0. 05 ） and TGF-β1 （ rmRNA = 0. 51, p 〈 0. 05 ）. There was positive correlation between the expression of TGF-β1 and of P27Kip1 （ rmRNA =0. 31, p 〈0. 05）. It is concluded that the apoptosis of APL cells is induced by As2O3 , and the cell cycle is arrested at G1 phase, The expression of P27^Kipl is closely related to the extent of apoptosis induced by As2O3. Apoptosis of APL cells induced by As2O3 may be caused by up-regulating TGF-β1 and P27Kip1 , which is antagonistic to cyclin E and BLC-2, leading to arrest of cell cycle at G1 phase.