3-MA对三氧化二砷诱导Jurkat细胞凋亡作用的影响

摘要

目的：探讨自噬特异性抑制剂3-甲基腺嘌呤（3-MA）对三氧化二砷（As2O3）诱导急性T细胞白血病系Jurkat细胞系Jurkat细胞凋亡的影响及机制。方法：XTT法检测三氧化二砷（ As2 O3）对急性T细胞白血病细胞系Jurkat细胞的增殖抑制作用。电镜下观察不同浓度As2 O3作用Jurkat细胞24 h后细胞形态。免疫印迹和流式细胞术检测微管相关蛋白1轻链3B（LC-3B）蛋白的表达变化。 AnnexinV-FITC/PI双染法流式细胞术检测3-MA对AS2O3诱导急性T细胞白血病系Jurkat细胞凋亡的影响。结果：As2 O3可以抑制急性T淋巴细胞白血病细胞株Jurkat细胞的生长，这种作用呈剂量和时间依赖性。2.5、5、10μmol/L AS2 O3作用Jurkat细胞24 h后在电镜下可观察到自噬、凋亡、坏死的不同形态，并且自噬体数目不断增多。5μmol/L As2 O3处理Jurkat细胞0、24、48 h后，LC-3B的平均荧光强度相对倍数分别（3.1±0.2）倍、（4.6±0.31）倍、（34.2±4.5）倍，组间相比，差异有统计学意义（P＜0.05），与生长抑制率一样呈现时间依赖性增强；免疫印迹也显示As2O3处理24 h和48 h后，LC-3B的蛋白表达逐渐增强；相对于As2O3组（33.4±9.1）％的生长抑制率，3-甲基腺嘌呤（3-MA）联合As2O3处理细胞后，48 h的生长抑制率为（60.6±8.3）％，差异明显，而LC-3B的表达明显降低。联合应用自噬抑制剂3-MA后Jurkat细胞的凋亡率（44.96±3.60）％，与单用As2 O3组（2.94±0.26）％相比明显增加，差异有统计学意义。结论：自噬抑制剂3-MA可以增加As2 O3引起的急性T细胞白血病细胞系Jurkat细胞的凋亡细胞百分数，其作用机制与诱导凋亡及抑制自噬密切相关。%Objective:To discuss the effect and mechanism of autophagy inhibitor 3-MA on arsenic trioxide inducing apoptosis of acute T-cell leukemia cell line Jurkat cells.Methods:Proliferation inhibition of Jurkat cells treated with arsenic trioxide was detected by XTT.Morphological characteristics of Jurkat cells treated with different concentrations arsenic trioxide were observed by electron mi-croscope.Microtubule-associated protein 1 light chain 3B (LC-3B) protein expression was detected by Western blot and flow cytome-try.Apoptosis rates of Jurkat cells treated with 3-MA combining arsenic trioxide were detected by flow cytometry using AnnexinV-FITC/PI double staining.Results:Arsenic trioxide inhibited the growth of Jurkat cells in a dose and time dependence.We observed different morphological characteristics of autophagy , apoptosis and necrosis accompanying more autophagosomes in Jurkat cells which were treated with arsenic trioxide 2.5,5,10 μmol/L after 24 h.LC3B mean fluorescence intensity (MFI)relative multiples were(3.1±0.2) fold,(4.6±0.31)fold,(34.2±4.5)fold with 5 μmol/L arsenic trioxide treated Jurkat cells 0,24,48 h,and the P values between each of the two groups were less than 0.05,which increased depending time consistently with the growth inhibition rates.LC-3B protein expression gradually increased treated Jurkat cells with arsenic trioxide after 24 h,48 h.The growth inhibition rate (60.6±8.3)%was significantly different treated with arsenic trioxide combining 3-methyl adenine ( 3-MA ) while it was ( 33.4 ±9.1 )% treated with arsenic trioxide alone, however, LC-3B protein expression gradually decreased.Jurkat cell apoptosis rate ( 44.96 ±3.60 )% was significantly increased treated with arsenic trioxide combining autophagy inhibitor(3-MA) while it was (2.94±0.26)% treated with arsenic trioxide alone, and this difference was statistically significant.Conclusion: 3-MA increased apoptosis rates of Jurkat cells inducing by Arsenic trioxide and it may be related with inhibition of autophagy and induction of apoptosis.