氨甲喋呤对映体药物对A549人肺腺癌细胞株增殖抑制作用及诱导凋亡作用

Effects of Methotrexate Enantiomers on Human Lung Cancer A549 Cells Inhibition and Its Apoptosis

关键词:  氨甲喋呤  对映体药物  A549细胞  MTT比色法  流式细胞术  DNA梯度电泳

Key words:  methotrexate  optical isomer  A549 cell  MTT  flow cytometry  DNA ladder

摘要:

目的 探讨氨甲喋呤对映体[(+)MTX，(-)MTX]对A549细胞的增殖抑制作用及诱导凋亡作用。方法 采用培养的A549细胞，应用MTT比色法分析其活性；用光学显微镜和荧光显微镜观察细胞的形态学变化；碘化丙啶(PI)单染流式细胞术检测细胞周期；DNA梯度电泳检测凋亡。结果 在0.1~150 μmol·L^-1范围内，(+)MTX和(-)MTX作用于A549细胞24，48，72 h，均抑制细胞A549增值，但抑制强度为(+)MTX>(-)MTX，倒置显微镜和荧光显微镜观察不同浓度(+)MTX和(-)MTX作用A549细胞不同时间后，出现细胞不同程度的形态学改变；用10 μmol·L^-1的(+)MTX和(-)MTX作用A549细胞48 h后，PI单染流式细胞术检测A549细胞周期的影响，表明氨甲喋呤对映体干扰A549细胞DNA合成；DNA梯度电泳检测结果发现MTX作用组有凋亡条带出现，其中(+)MTX最为明显。结论 (+)MTX和(-)MTX对A549细胞的抗增殖作用具有化学结构的立体选择性，(+)MTX的抗A549细胞增殖作用明显强于(-)MTX。

Abstract:

OBJECTIVE To investigate the effect of methotrexate enantiomers [included (+)MTX, (-)MTX] on the proliferation and apoptosis of A549 cells. METHODS A549 cells were cultured. The cell proliferation was determined by MTT. The morphological changes were inspected by inverted microscope and fluorescence microscope. Cell cycle phases were assayed by propidium iodide staining flow cytometry. DNA ladder were used to detect the apoptosis. RESULTS A549 cells were treated with (+)MTX and (-)MTX at 1-150 μmol·L^-1 for 24, 48, 72 h. The results showed that the proliferations of A549 cells were significantly inhibited under the different conditions. The order of the inhibited efficacy was (+)MTX>(-)MTX. The morphological of A549 cells were found changes by (+)MTX and (-)MTX treatment. After administration of 10 μmol·L^-1 of (+)MTX and (-)MTX for 48 h, respectively, the cell cycle phases were assayed by propidium iodide staining flow cytometry. DNA ladder was the most recognized marker of apoptosis, and there was obvious DNA ladder in (+)MTX treated group. The result showed DNA replication was interfered by (+)MTX and (-)MTX treatment. CONCLUSION The proliferation of A549 cells have the chiral selective effects by (+)MTX and (-)MTX treatment, and the inhibition on A549 cells proliferation of (+)MTX was significantly stronger than (-)MTX.