【作者】 郭立方；

【导师】 贾正平； 王荣；

【作者基本信息】 兰州大学 ， 药理学， 2010， 硕士

【摘要】 1.探讨氨甲喋呤对映体((+)MTX、(-)MTX)对ECV304,A549, HepG2细胞的增殖抑制作用及诱导凋亡实验研究。2.快速灵敏的液相色谱串联质谱法测定氨甲喋呤对映体作用HepG2后,药物在细胞内外液中的浓度。方法1.采用培养的ECV304, HepG2, A549细胞,应用MTT比色法分析MTX对映体的活性。2.用倒置显微镜观察ECV304, HepG2, A549细胞的形态学变化。3.碘化丙啶(PI)单染流式细胞术检测ECV304, HepG2, A549的细胞周期。4.荧光显微镜DAPI观察A549细胞的形态学变化及凋亡。5.DNA梯度电泳检测细胞凋亡。6.异硫氰酸荧光素／碘化丙啶(V-FITC／PI)双染流式细胞术检测HepG2凋亡7.液相色谱串联质谱法测定MTX对映体在细胞内外液中的浓度。结果1.在0.1,umol·L-1-150μmol·L-1范围内,(+)MTX和(-)MTX作用于ECV304, HepG2,A549细胞24、48、72h,均抑制细胞ECV304, HepG2, A549增值,但抑制强度为(+)MTX>(-)MTX。2.倒置显微镜观镜察不同浓度(+)MTX和(-)MTX作用ECV304, HepG2, A549细胞不同时间后,出现细胞不同程度的形态学改变,且(+)MTX作用的细胞形态学改变强于(-)MTX。3.(+)MTX和(-)MTX作用ECV304, HepG2和A549细胞,PI单染流式细胞术检测ECV304, HepG2, A549细胞周期的影响,表明氨甲喋呤对映体均干扰ECV304, HepG2,A549细胞DNA合成且出现不同程度的凋亡峰。4.荧光显微镜DAPI染色察不同浓度(+)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,有凋亡小体产生。5.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。6.(+)MTX和(-)MTX作用HepG2细胞后,细胞出现不同程度的凋亡。7.(+)MTX和(-)MTX作用HepG2细胞后,测得(+)MTX在细胞内浓度高于(-)MTX。结论(+)MTX和(-)MTX作用ECV304, HepG2, A549细胞后,能够诱导细胞凋亡,且(+)MTX和(-)MTX对ECV304, HepG2, A549细胞的抗增殖作用具有化学结构的立体选择性,(+)MTX的抗细胞增殖作用明显强于(-)MTX。更多还原

【Abstract】 8Objective:1.To investigate the effect of MTX(included (+)MTX and (-)MTX)on the proliferation of ECV304 cells and to explore its mechanisms.2. A rapid and sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS/MS) was developed and validated for the quantitative determination the concentration of methotrexate (MTX) enantiomers in the intracellular and extracellular fluid.Methods:1.ECV304, HepG2, A549 cells were cultured. The cell proliferation was determined by MTT.2. The morphological change of ECV304, HepG2, A549 cells were inspected by inverted microscope.3.Cell cycle phases of ECV304, HepG2, A549 were assayed by propidium iodide staining flow cytometry.4. Fluorescentm micrographs of A549 induced with (+)MTX, (-)MTX indicated by DAPI.5.DNA ladder were used to detect the apoptosis.6. Cell apoptosis of was determined by Annexin V-FITC (fluoresceinisothiocyanate)/PI (propidium iodide) staining.7. Liquid chromatography-tandem mass spectrometry assay (LC-MS/MS)with electrospray ionization was developed and validated for the quantitative determination the concentration of methotrexate enantiomers in the intracellular and extracellular fluid. Results:1.ECV304, HepG2 and A549 cells were treated with (+)MTX, (-)MTXat 1μmiol·L-150μmol·L-1 for 24h,48h,72h. The results showed that the proliferation of ECV304, HepG2 and A549 cells were significantly inhibited under the different conditions. The order of the inhibited efficacy was(+)MTX>(-)MTX.2. The Morphological of ECV304, HepG2 and A549 cells were found to changes by (+)MTX and(-)MTX treatment.3.After administration of 10μmol·L-1 of (+)MTX, (-)MTX for 48h, the cell cycle phases were assayed by propidium iodide staining flow cytometry. The result showed DNA replication was interfered by (+)MTX and (-)MTX treatment.4. The Morphological of A549 cells were found to changes by(+)MTX and (-)MTX treatment that included the cell shrinkage, chromatin condensation and apoptosis body.5.DNA ladder was the most recognized marker of apoptosis, and there was obvious DNA ladder in (+)MTX treated group.6. DNA ladder was the most recognized marker of apoptosis, and there was obvious DNA ladder in (+)MTX treated group.7.The different concentrations of MTX enantiomers in the intracellular and extracellular of HepG2 cells were obtained.It were shown that (+)MTX concentration much higher than (-)MTX concentration in intracellular of HepG2 cells.Conclusion:The proliferation of ECV304, HepG2 and A549 cells have the chiral selective effects by (+)MTX and(-)MTX treatment, and the inhibition on ECV304, HepG2 and A549 cells proliferation of (+)MTX was significantly stronger than (-)MTX.更多还原