三氧化二砷对Molt-4细胞株Notch信号通路作用机制的研究

Effect of arsenic trioxide on Notch1 gene in acute lymphoblastic leukemia cells

目的 :探讨三氧化二砷(As2O3)对急性淋巴细胞白血病细胞株Molt-4中Notch1基因的作用机制。方法 :采用反转录(RT)-PCR检测正常人外周血淋巴细胞与急性淋巴细胞白血病细胞株A3和Molt-4中Notch1基因m RNA的相对表达量,选取Notch1 m RNA表达量较高的Molt-4细胞,分别经浓度0、2、4μmol/L As2O3处理细胞后,应用细胞计数试剂盒(CCK8)检测细胞活力,显微镜下观察细胞形态学变化,流式细胞仪检测细胞凋亡率,RT-PCR检测Notch1 m RNA表达率,蛋白质印迹法检测细胞内Notch1、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)及胱天蛋白酶3(caspase-3)凋亡蛋白的表达情况。结果:As2O3能下调Molt-4细胞中Notch1基因及蛋白的表达,降低Bcl-2/Bax蛋白比例,并促进凋亡蛋白胱天蛋白酶3的活化,2μmol/L As2O3处理后,随作用时间延长,Molt-4细胞的凋亡率逐渐增高(r=0.989,P<0.05)。Molt-4细胞分别经2μmol/L和4μmol/L As2O3处理72 h后,流式结果显示,随As2O3浓度升高,Molt-4细胞凋亡率升高(r=1.000,P<0.05)。As2O3以时间和浓度依赖的方式诱导Molt-4细胞凋亡。结论:As2O3通过抑制Molt-4细胞株Notch1信号通路的表达,抑制其增殖并诱导凋亡。

Objective To investigate the effect of arsenic trioxide（As2O3） on Notch1 gene in acute lymphoblastic leukemia Molt-4 cell line. Methods Reverse transcription polymerase chain reaction（RT-PCR） was used to detect the expression of Notch1 m RNA in peripheral blood mononuclear cells of healthy adult and two acute lymphoblastic leukemia cell lines： A3 and Molt-4 cells. Molt-4 cells which had higher expression of Notch1 m RNA were selected as the research object. Molt-4 cells were treated with different concentrations of As2O3（0, 2, and 4 μmol/L） for 24, 48 and 72 h. Then were tested for cell viability by cell counting kit-8（CCK-8）, cell morphology was observed by microscope and flow cytometry was used to assess apoptosis. Notch1 m RNA expression was detected by quantitative PCR（q PCR）; Notch1, B cell lymphoma/leukemia-2（Bcl-2）, Bcl-2-associated X protein（Bax） and caspase-3 protein were determined by Western blot.Results Treatment with 2 μmol/L As2O3 for 48 and 72 h, the apoptosis of Molt-4 cells increased gradually（r=0.989, P〈0.05）. When Molt-4 cells were treated with 2 μmol/L and 4 μmol/L As2O3 for 72 h,the apoptosis of Molt-4 cells increased gradually（r=1.000, P〈0.05）. The results demonstrated that As2O3 inhibited cell proliferation and induced apoptosis in a time and concentration dependent manner by decreasing the expression of Notch1, reducing the ratio of Bcl-2/Bax, as well as increasing activation of caspase-3 in Molt-4 cells. Conclusions As2O3 inhibits acute lymphoblastic leukemia cell growth and induces apoptosis through inactivation of Notch signaling pathway.