As_2O_3在HuH7及CD13^+CD133^+LCSCs增殖和分化中作用的初步观察  

Role of arsenic trioxide in proliferation of HuH7 cells and differentiation of CD13^+CD133^+LCSCs

作　　者：金世龙[1] 余天雾[1] 黄中荣[1] 曹洪[1] 龙运全[1] 周健[1] 李鹤[1] 苟毅[1] 廖娟[1] 李远[2] 

机构地区：[1]重庆医科大学附属永川医院肝胆外科,重庆402160 [2]重庆医科大学附属永川医院中心实验室,重庆402160

出　　处：《第三军医大学学报》2012年第18期1857-1861,共5页Journal of Third Military Medical University

摘　　要：目的观察As2O3对人肝癌细胞系HuH7及CD13+CD133+肝癌干细胞(liver cancer stem cells,LCSCs)增殖和分化的作用。方法用荧光激活流式细胞术(fluorescence activated cell sorter,FACS)从HuH7细胞系分选CD13+CD133+LCSCs,MTT和EdU法分析As2O3刺激HuH7及CD13+CD133+LCSCs增殖作用。流式细胞术检测As2O3处理第3、5天HuH7细胞凋亡,和As2O3处理第3天细胞周期变化。Western blot分析HuH7粒细胞白血病(promyelocytic leukaemia,PML)蛋白表达情况。观察As2O3处理前后HuH7对吡柔比星敏感性和肿瘤球形成能力变化。结果早期(5 d)低浓度As2O3促进HuH7和CD13+CD133+LCSCs增殖,高浓度As2O3则抑制CD13+CD133+LCSCs增殖。As2O3虽然没有阻滞HuH7细胞周期,但使G0/G1期细胞降低2.32%。As2O3处理3 d的HuH7细胞凋亡与正常对照HuH7细胞相似,凋亡率在8%～9.5%,第5天使HuH7细胞凋亡率上升到12%～15%,1.0μg/ml As2O3组晚期凋亡率比0.5μg/ml As2O3高。As2O3改变HuH7细胞对THP敏感性,还显著降低CD13+CD133+LCSCs成球能力。HuH7细胞表达PML蛋白,As2O3可使HuH7细胞PML蛋表达降低,而且与HuH7细胞和CD13+CD133+LCSCs增殖有刺激作用,HuH7细胞凋亡及CD13+CD133+LCSCs分化结果一致。结论低浓度As2O3不仅刺激HuH7及CD13+CD133+LCSCs增殖,促进HuH7细胞凋亡,而且可能诱导CD13+CD133+LCSCs分化,其作用通过PML蛋白介导。Objective To determine the effect of arsenic trioxide （As2O3 ） on the proliferation and differentiation of human liver cancer cell line HuH7 and its CD13 ＋ CD133＋liver cancer stem cells （LCSCs）. Methods CD13＋ CD133 ＋ LCSCs was sorted from HuH7 cells by fluorescence activated cell sorter （FACS）. MI3＋ assay and EdU incorporation was used to analyze As2O3 stimulation in the proliferation of HuH7 cells and CD13 ＋ CD133 ＋ LCSCs. FACS was used to detected HuH7 cells＇ apoptosis on the third day and fifth day stimu- lated by As2O3, and HuH7 cell circle on the third day. Expression of promyelocytic leukaemia （PML） protein was detected by Western blotting. The sensitivity of HuH7 cells to pirarubicin （THP） and formation of tumor sphere was observed. Results As2O3 at low concentration induced the proliferation in HuH7 cells and CD13＋ CD133＋LCSCs in the early stage （within 5 d）, and that at high concentration suppressed CD13 ＋ CD133＋ LCSCs proliferation. Although As2O3 did not block the circle of HuH7 cells, it had reduced cells at G0/G1 phase by 2.32%. After 3 days＇ treatment of As2O3, the apoptotic rate of HuH7 cells was 8% to 9.5%, similar to the cells without treatment, but the rate was increased to 12% to 15% in 5 d after treatment. As2O3 of 1. 0μg/ml resulted a higher apoptotic rate than that of 0.5 μg/ml in the late stage. As2O3 enhanced HuH7 cells＇ sensitivity to THP, and reduced CD13＋ CD133＋ LCSCs＇ capacity to form tumor sphere. PML protein was expressed in the HuH7 cells, which was down-regulated by As2O3. Conclusion Low concentration As203 enhances the proliferation of HuH7 cells and CD13＋ CD133＋ LCSCs, induces apoptosis in HuH7 cells, and improves CD13＋ CD133＋ LCSCs differentiate. The effects might be mediated by PML.

关 键 词：肝癌 癌干细胞 早幼粒细胞白血病蛋白 三氧化二砷 分化 

分 类 号：R735.7[医药卫生—肿瘤] R965[医药卫生—临床医学]