氨甲喋呤对映体对细胞的抑制作用及机制研究
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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。
8
Objective:
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.
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。
8
Objective:
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.
引文
[1]卢海刚,赵丁,任雷鸣.多沙唑嗪对映体对兔离体膀胱逼尿肌的作用及机制[J].中国药理学通报,2008,24(4)：513-517.
[2]王荣英,任雷鸣.多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖的选择性抑制作用[J].中国药理学通报,2009,25(1)：81-84.
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[8]董林,何晓东,陶绍能,等.甲氨蝶呤对映体诱导的肺癌A549耐药细胞株中VEGF及其受体表达差异的研究[J].肿瘤,2009,5(29)404-408.
[9]Kaufmann S.Induction of endonucleolytic DNA cleavage in human acutemyelogenous leukemia cells by etoposide, captothecin and the other cytotoxic anticancer drugs:a cautionary note[J].Cancer Res,1989,4 (9):5870-5878.
[10]Cutolo M, Bisso A, Sulli A, et al. Antiproliferative and antiinflammatory effect of methotrexate on cultured differentiating myeloid monocytic cells but not on synovial macrophages from patients with rheumatoid arthritis[J].Rheumatol, 2000,2(7):2551-2557.
[11]Merkle CJ, Moore IM, Penton BS,et al. Methotrexate causes apoptosis in postmitotic endothelial cells[J].Biol.Res. Nurs,2000,2(1):4-14.
[12]Bokhari M, Carnachan RJ, Cameron NR, et al. Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge[J].Anat,2007,21(1):567-576.
[13]Folkman J, Browder T, Palmblad J.Angiogenesis research:guide lines for translation to clinical application [J].Thromb Haemost,2001,86(1):23-33.
[14]王荣,贾正平,陈立仁,等.毛细管电泳对氨甲喋呤对映体拆分及人血样中对映体含量测定研究[J].分析测试学报,2005,24(2)：19-23.
[15]郭立方,王荣,贾正平,等.MTF分析法检测氨甲蝶呤对映对体对ECV304内皮细胞的研究.中国医院药学杂志.2009,29(20)：1701-1704.
[16]郭立方,王荣,贾正平,等.氨甲喋呤对映体对ECV304细胞抑制作用及其机制研究.中国药理学通报2010,26(2)：213-216.
[1]卢海刚,赵丁,任雷鸣.多沙唑嗪对映体对兔离体膀胱逼尿肌的作用及机制[J].中国药理学通报,2008,24(4)：513-517.
[2]王荣英,任雷鸣.多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖的选择性抑制作用[J].中国药理学通报,2009,25(1)：81-84.
[3]孙云,谭文,周华珠,等.左旋沙丁胺醇抑制豚鼠气道平滑肌收缩的实验研究[J].中国药理学通报,2008,24(2)：196-208
[4]王荣,贾正平,陈立仁,等.毛细管电泳对氨甲喋呤对映体拆分及人血样中对映体含量测定研究[J].分析测试学报,2005,24(2)：19-23.
[5]Kaufmann S.Induction of endonucleolytic DNA cleavage in human acutemyelogenous leukemia cells by etoposide, captothecin and the other cytotoxic anticancer drugs:a cautionary note[J].Cancer Res,1989,4 (9):5870-5878.
[6]Cutolo M, Bisso A, Sulli A, Felli L, Briata M, Pizzorini C, Villaggio B. Antiproliferative and antiinflammatory effect of methotrexate on cultured differentiating myeloid monocytic cells but not on synovial macrophages from patients with rheumatoid arthritis[J].Rheumatol,2000,2(7):2551-2557.
[7]Merkle CJ, Moore IM, Penton BS, Torres BJ, Cueny RK, Schaeffer RC, Montgomery DW. Methotrexate causes apoptosis in postmitotic endothelial cells[J].Biol.Res.Nurs, 2000,2(1):4-14.
[8]Bokhari M, Carnachan RJ, Cameron NR, Przyborski SA. Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge[J].Anat,2007,21(1):567-576.
[9]董林,何晓东,陶绍能,等.甲氨蝶呤对映体诱导的肺癌A549耐药细胞株中VEGF及其受体表达差异的研究[J].肿瘤,2009,5(29)404-408.
[10]Folkman J, Browder T, Palmblad J.Angiogenesis research:guide lines for translation to clinical application [J].Thromb Haemost,2001,86(1):23-33.
[11]Itoh T, Ono K,Koido KI.et al.Stereoselectivity of the folate transporter in rabbit small intestine:studies with amethopterin enantiomers [J].Chirality,2001,13:164-169.
[12]Narawa T,Shimizu R,Takano S,et al.Stereoselectivity of the Reduced Folate Carrier in Caco-2 Cells[J].Chirality,2005,17:444-449.
[13]Magdalena Dabrowska, Grazyna Mosieniak, Janusz Skierski, et al. Methotrexat-induced senescence in human adenocarcinoma cells is accompanied by induction of p21waf1/cip1 expression and lack of polyploidy. Cancer Lett.,2009,284(1):95-101.
[14]I.W. Taylor, M.H.N. Tattersall. Methotrexate cytotoxicity in cultured human leukemic cells studied by flow cytometry, Cancer Res,1981,41:1549-1556.
[1]黄晓龙.美国FDA关于开发立体异构体新药的政策简介[J].中国新药杂志.2000,9(9)：650-652.
[2]郭立方,王荣,贾正平,等.氨甲喋呤对映体对ECV304细胞抑制作用及其机制研究[J].中国药理学通报,2010,26(2)：213-216.
[3]王荣,贾正平,陈立仁,等.毛细管电泳对氨甲喋呤对映体拆分及人血样中对映体含量测定研究[J].分析测试学报,2005,24(2)：19-23.
[4]Treon S P,Chabner B A.. Concepts in use of high-dose methotrexate therapy. Clin Chem,1996,42:1322-1329.
[5]TAO S N, HE X D,DONG L, LI M, ZHU Y Y, SUN Z M, SHEN Z. Establishment of Methotrexate Enantiomers Resistant A549 Cell Lines of Human NSCCL and Its Biological Characteristics[J].Cancer Research on Prevention and Treatment.2009,36 (4):273-277
[6]董林,何晓东,陶绍能,等.甲氨蝶呤对映体诱导的肺癌A549耐药细胞株中VEGF及其受体表达差异的研究[J].肿瘤,2009,5(29)404-408.
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[9]Belkov V M, Krynetski E Y, Schuetz J D,et al.Reduced folate carrier expression in acute lymphoblastic leukemia:a mechanism for ploidy but not lineage differences in methotrexate accumulation[J].Blood,1999,9(2):1643-1650.
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[11]Walker T M, Rhodes P C, Westmoreland C, The differential cytotoxicity of methotrexate in rat hepatocyte monolayers and spheroid cultures, Toxicol[J].In Vitro, 2000,14:475-485.
[12]Bokhari M, Carnachan R J, Cameron N R, Przyborski S A. Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge[J].Anat,2007,211:567-576.
[13]Youn-Jung Kim, Mee Song. Inflammation in methotrexate-induced pulmonary toxicity occurs via the p38 MAPK pathway[J].Toxicology,2009,256:83-190.
[14]Ya-Xia Chen.Methotrexate induces apoptosis of human choriocarcinoma cell line JAR via a mitochondrial pathway[J].European Journal of Obstetrics & Gynecology and Reproductive Biology,2009,143:107-111.
[15]Itoh T, Ono K,Koido KI.et al. Stereoselectivity of the folate transporter in rabbit small intestine:studies with amethopterin enantiomers [J].Chirality,2001,13:164-169.
[16]Narawa T,Shimizu R,Takano S.et al.Stereoselectivity of the Reduced Folate Carrier in Caco-2 Cells[J].Chirality,2005,17:444-449.
[17]Magdalena Dabrowska, Grazyna Mosieniak, Janusz Skierski,et al.Methotrexate-induced senescence in human adenocarcinoma cells is accompanied by induction of p21wafl/cipl expression and lack of polyploidy[J].Cancer Lett.,2009,284(1):95-101.
[1]傅崇东,徐惠南.手性药物的立体选择性药物动力学[J].中国临床药学杂志,1998,7(6)：309-313.
[2]黄晓龙.美国FDA关于开发立体异构体新药的政策简介[J].中国新药杂志.2000,9(9)：650-652.
[3]李高.手性药物的临床药物动力学立体选择性[J].中国医院药学杂志,1999,19(7)：429-431.
[4]Meyer MC, Guttman DE. The binding of drugs by plasma proteins [J].Journal of Pharmaceutical Sciences,1968,57(6):895-899.
[5]Kaufmann S.Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, captothecin and the other cytotoxic anticancer drugs:a cautionary note[J].Cancer Res.,1989,4(9):5870-5879.
[6]Cutolo M, Bisso A, Sulli A, Felli L, Briata M, Pizzorini C, Villaggio B. Antiproliferative and antiinflammatory effect of methotrexate on cultured differentiating myeloid monocytic cells but not on synovial macrophages from patients with rheumatoid arthritis[J].Rheumatol,2000,2(7),2551-2557.
[7]Merkle CJ, Moore IM, Penton BS, Torres BJ, Cueny RK, Schaeffer RC, Montgomery DW. Methotrexate causes apoptosis in postmitotic endothelial cells [J].Biol. Res. Nurs., 2000.2(1),4-10.
[8]Bokhari M, Carnachan RJ,Cameron NR, Przyborski SA. Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge [J].Anat.,2007,21(1),567-569.
[9]王荣,贾正平,陈立仁,等.毛细管电泳对氨甲喋呤对映体拆分及人血样中对映体含量测定研究[J].分析测试学报,2005,24(2)：19-23.
[10]Itoh T, Ono K, Koido KI.et al. Stereoselectivity of the folate transporter in rabbit small intestine:studies with amethopterin enantiomers [J].Chirality,2001,19(3):164-169.
[11]Masson E,Relling MV,Synold TW, et al.Accumulation of methotremate polyglutamates in lymphoblasts is a determinant of antileukemic effects in vivo [J].Clin Inves.,1996,9(7):73-78.
[12]Longo GS,Gorlick R, Tong WP, et al.Disparate affinities of antifolates or folylpolyglutamate synthetase from human leukemia cell [J].Blood,1997,9 (4):1241-1247.
[1]傅崇东,徐惠南.手性药物的立体选择性药物动力学[J].中国临床药学杂志,1998,7(6)：309-313.
[2]黄晓龙.美国FDA关于开发立体异构体新药的政策简介[J].中国新药杂志.2000,9(9)：650-652.
[3]李高.手性药物的临床药物动力学立体选择性[J].中国医院药学杂志,1999,19(7)：429-431.
[4]Meyer MC, Guttman DE. The binding of drugs by plasma proteins [J].Journal of Pharmaceutical Sciences,1968,57(6):895-899.
[5]Kaufmann S.Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, captothecin and the other cytotoxic anticancer drugs:a cautionary note[J].Cancer Res.,1989,4 (9):5870-5879.
[6]Cutolo M, Bisso A, Sulli A, Felli L, Briata M, Pizzorini C, Villaggio B. Antiproliferative and antiinflammatory effect of methotrexate on cultured differentiating myeloid monocytic cells but not on synovial macrophages from patients with rheumatoid arthritis[J].Rheumatol,2000,2(7),2551-2557.
[7]Merkle CJ, Moore IM, Penton BS,Torres BJ,Cueny RK, Schaeffer RC, Montgomery DW. Methotrexate causes apoptosis in postmitotic endothelial cells [J].Biol. Res. Nurs.,2000.2(1),4-10
[8]Bokhari M, Carnachan RJ, Cameron NR, Przyborski SA. Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge [J].Anat.,2007,21(1),567-569.
[9]王荣,贾正平,陈立仁,等.毛细管电泳对氨甲喋呤对映体拆分及人血样中对映体含量测定研究[J].分析测试学报,2005,24(2)：19-23.
[1]Amato I.Looking glass chemistry[J].Science,1992,256:964-968.
[2]于平.手性化合物研究进展[J].化工进展,2002,21(9)：536-538.
[3]官家乐,高原,高鸿慈.手性药物[J].中国医院药学杂志,2001,21(7)：443-444..
[4]宫丽,卞手性药物的药理学立体选择性[J].国外医学药学分册,2007,34(2)：321-324.
[5]何凤慈,陈亮,蒋耀光.酮洛芬及其手性对映体的研究进展[J].中国药房,2004,15(4)：244-246.
[6]杜玉民,刘伟娜,白希瑞.手性药物[J].临床荟萃,2003,18(20)：6914-6916.
[7]王丹,何浪,手性与手性药物[J].成都医学院学报,2006,1(2)：155-147.
[8]陈亮,何凤慈.光学活性药物的药动学和药效学差异[J].中国新药与临床杂志,2003,22(6)：377-380.
[9]蒋兆健,吴笑春.手性药物的对映体选择性与临床应用[J].中国药房,2001,12(3)：162-164.
[10]李良智.史大永,刘均洪,等.手性药物的构型与药效活性[J].青岛化工学院学报,2001,22(2)：124-126.
[11]王普善,王宇梅.手性药物开发战略的再认识[J].精细与专用化学品,2004,12(10)：4-8.
[12]Caldwell J.The importance of stereochemistry in drug action and disposition.J Clin Pharmacol,1992,32:925-927.
[13]Hutt A,Tan SC.Drug chirality and its clinical significance.Drugs,1996,52(5):1-4.
[141曾苏,沈向忠,刘志强.手性药物的药理及其体内对映体的HPLC测定.浙江医科大学学报.1994,23(5)：234-236.
[15]王似菊,沈向忠,曾苏.心血管药物的立体选择性活性及临床意义.中国医院药学杂志,1995,15(6)：284-286.
[16]Peng SX, Henson C,WilsonLJ.Sinultancous determination of enantioselective plasma protein binding of aminohydantoins by ultrafiltration and chiral hiph-performance liquid chromatography[J].Chromatogr B Biomed Sci Appl.1999;73:31-37.
[17]Menard C, Lamiable D, Vistelle R, et al. Stereoselective Biotransfomation of cicletamine in cultured rat and human hepatocytes[J].Pharmacol Res,2004;42(1):87-92.
[18]Drayer DE.Problems in therapeutic drug monitoring:the dilemma of enantiomeric drugs in man.The Drug Monitoring,1988,10(1):1-5.
[19]Zeng S,Zhang L,Liu ZQ,et al.Stereoselective metabolism of ofloxacin in human.1995, 9(1):87-90.
[20]Jamali F,Mehvar R,Pasutto.Enantioselective aspects of drug action and disposition: therapeuticpitfalls.J Pharm Sci,1989,78(9):695-699.
[21]Lee EJD,Williams KM.Chirality clinical pharmacokinetic and pharmacodynamic consideration.Clin Pharmacokinet,1990,18(2):339-343..
[22]段立华.多沙唑嗪对映体对大鼠离体心肌和豚鼠离体气管平滑肌的作用[D].河北医科大学,2007,9-14.
[23]毛小琴,邱宗荫,邱峰,等.普萘洛尔对映异构体诱导HUVEC细胞的蛋白质表达谱差异.中国生物化学与分子生物学报,2008,24(2)：165-171.
[2]王荣英,任雷鸣.多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖的选择性抑制作用[J].中国药理学通报,2009,25(1)：81-84.
[3]Peng SX, HensonC, WilsonLJ.Sinultancous determination of enantioselective plasma protein binding of aminohydantoins by ultrafiltration and chiral hiph-performance liquid chromatography[J].J Chromatogr B Biomed Sci Appl.1999;73:31-37.
[4]Menard C, Lamiable D, Vistelle R, et al. Stereoselective Biotransfomation of cicletamine in cultured rat and human hepatocytes[J].Pharmacol Res,2004;42(1):87-92.
[5]曾苏,李艳,王似菊.新药开发中的手性与临床药代动力学因素.中国医药工业杂志,1996,27(9)：430-438.
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[7]Treon SP, Chabner B A. Concepts in use of high2dose methotrexate therapy. Clin Chem,1996,42:1322-1329.
[8]董林,何晓东,陶绍能,等.甲氨蝶呤对映体诱导的肺癌A549耐药细胞株中VEGF及其受体表达差异的研究[J].肿瘤,2009,5(29)404-408.
[9]Kaufmann S.Induction of endonucleolytic DNA cleavage in human acutemyelogenous leukemia cells by etoposide, captothecin and the other cytotoxic anticancer drugs:a cautionary note[J].Cancer Res,1989,4 (9):5870-5878.
[10]Cutolo M, Bisso A, Sulli A, et al. Antiproliferative and antiinflammatory effect of methotrexate on cultured differentiating myeloid monocytic cells but not on synovial macrophages from patients with rheumatoid arthritis[J].Rheumatol, 2000,2(7):2551-2557.
[11]Merkle CJ, Moore IM, Penton BS,et al. Methotrexate causes apoptosis in postmitotic endothelial cells[J].Biol.Res. Nurs,2000,2(1):4-14.
[12]Bokhari M, Carnachan RJ, Cameron NR, et al. Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge[J].Anat,2007,21(1):567-576.
[13]Folkman J, Browder T, Palmblad J.Angiogenesis research:guide lines for translation to clinical application [J].Thromb Haemost,2001,86(1):23-33.
[14]王荣,贾正平,陈立仁,等.毛细管电泳对氨甲喋呤对映体拆分及人血样中对映体含量测定研究[J].分析测试学报,2005,24(2)：19-23.
[15]郭立方,王荣,贾正平,等.MTF分析法检测氨甲蝶呤对映对体对ECV304内皮细胞的研究.中国医院药学杂志.2009,29(20)：1701-1704.
[16]郭立方,王荣,贾正平,等.氨甲喋呤对映体对ECV304细胞抑制作用及其机制研究.中国药理学通报2010,26(2)：213-216.
[1]卢海刚,赵丁,任雷鸣.多沙唑嗪对映体对兔离体膀胱逼尿肌的作用及机制[J].中国药理学通报,2008,24(4)：513-517.
[2]王荣英,任雷鸣.多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖的选择性抑制作用[J].中国药理学通报,2009,25(1)：81-84.
[3]孙云,谭文,周华珠,等.左旋沙丁胺醇抑制豚鼠气道平滑肌收缩的实验研究[J].中国药理学通报,2008,24(2)：196-208
[4]王荣,贾正平,陈立仁,等.毛细管电泳对氨甲喋呤对映体拆分及人血样中对映体含量测定研究[J].分析测试学报,2005,24(2)：19-23.
[5]Kaufmann S.Induction of endonucleolytic DNA cleavage in human acutemyelogenous leukemia cells by etoposide, captothecin and the other cytotoxic anticancer drugs:a cautionary note[J].Cancer Res,1989,4 (9):5870-5878.
[6]Cutolo M, Bisso A, Sulli A, Felli L, Briata M, Pizzorini C, Villaggio B. Antiproliferative and antiinflammatory effect of methotrexate on cultured differentiating myeloid monocytic cells but not on synovial macrophages from patients with rheumatoid arthritis[J].Rheumatol,2000,2(7):2551-2557.
[7]Merkle CJ, Moore IM, Penton BS, Torres BJ, Cueny RK, Schaeffer RC, Montgomery DW. Methotrexate causes apoptosis in postmitotic endothelial cells[J].Biol.Res.Nurs, 2000,2(1):4-14.
[8]Bokhari M, Carnachan RJ, Cameron NR, Przyborski SA. Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge[J].Anat,2007,21(1):567-576.
[9]董林,何晓东,陶绍能,等.甲氨蝶呤对映体诱导的肺癌A549耐药细胞株中VEGF及其受体表达差异的研究[J].肿瘤,2009,5(29)404-408.
[10]Folkman J, Browder T, Palmblad J.Angiogenesis research:guide lines for translation to clinical application [J].Thromb Haemost,2001,86(1):23-33.
[11]Itoh T, Ono K,Koido KI.et al.Stereoselectivity of the folate transporter in rabbit small intestine:studies with amethopterin enantiomers [J].Chirality,2001,13:164-169.
[12]Narawa T,Shimizu R,Takano S,et al.Stereoselectivity of the Reduced Folate Carrier in Caco-2 Cells[J].Chirality,2005,17:444-449.
[13]Magdalena Dabrowska, Grazyna Mosieniak, Janusz Skierski, et al. Methotrexat-induced senescence in human adenocarcinoma cells is accompanied by induction of p21waf1/cip1 expression and lack of polyploidy. Cancer Lett.,2009,284(1):95-101.
[14]I.W. Taylor, M.H.N. Tattersall. Methotrexate cytotoxicity in cultured human leukemic cells studied by flow cytometry, Cancer Res,1981,41:1549-1556.
[1]黄晓龙.美国FDA关于开发立体异构体新药的政策简介[J].中国新药杂志.2000,9(9)：650-652.
[2]郭立方,王荣,贾正平,等.氨甲喋呤对映体对ECV304细胞抑制作用及其机制研究[J].中国药理学通报,2010,26(2)：213-216.
[3]王荣,贾正平,陈立仁,等.毛细管电泳对氨甲喋呤对映体拆分及人血样中对映体含量测定研究[J].分析测试学报,2005,24(2)：19-23.
[4]Treon S P,Chabner B A.. Concepts in use of high-dose methotrexate therapy. Clin Chem,1996,42:1322-1329.
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[6]董林,何晓东,陶绍能,等.甲氨蝶呤对映体诱导的肺癌A549耐药细胞株中VEGF及其受体表达差异的研究[J].肿瘤,2009,5(29)404-408.
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[9]Belkov V M, Krynetski E Y, Schuetz J D,et al.Reduced folate carrier expression in acute lymphoblastic leukemia:a mechanism for ploidy but not lineage differences in methotrexate accumulation[J].Blood,1999,9(2):1643-1650.
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[11]Walker T M, Rhodes P C, Westmoreland C, The differential cytotoxicity of methotrexate in rat hepatocyte monolayers and spheroid cultures, Toxicol[J].In Vitro, 2000,14:475-485.
[12]Bokhari M, Carnachan R J, Cameron N R, Przyborski S A. Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge[J].Anat,2007,211:567-576.
[13]Youn-Jung Kim, Mee Song. Inflammation in methotrexate-induced pulmonary toxicity occurs via the p38 MAPK pathway[J].Toxicology,2009,256:83-190.
[14]Ya-Xia Chen.Methotrexate induces apoptosis of human choriocarcinoma cell line JAR via a mitochondrial pathway[J].European Journal of Obstetrics & Gynecology and Reproductive Biology,2009,143:107-111.
[15]Itoh T, Ono K,Koido KI.et al. Stereoselectivity of the folate transporter in rabbit small intestine:studies with amethopterin enantiomers [J].Chirality,2001,13:164-169.
[16]Narawa T,Shimizu R,Takano S.et al.Stereoselectivity of the Reduced Folate Carrier in Caco-2 Cells[J].Chirality,2005,17:444-449.
[17]Magdalena Dabrowska, Grazyna Mosieniak, Janusz Skierski,et al.Methotrexate-induced senescence in human adenocarcinoma cells is accompanied by induction of p21wafl/cipl expression and lack of polyploidy[J].Cancer Lett.,2009,284(1):95-101.
[1]傅崇东,徐惠南.手性药物的立体选择性药物动力学[J].中国临床药学杂志,1998,7(6)：309-313.
[2]黄晓龙.美国FDA关于开发立体异构体新药的政策简介[J].中国新药杂志.2000,9(9)：650-652.
[3]李高.手性药物的临床药物动力学立体选择性[J].中国医院药学杂志,1999,19(7)：429-431.
[4]Meyer MC, Guttman DE. The binding of drugs by plasma proteins [J].Journal of Pharmaceutical Sciences,1968,57(6):895-899.
[5]Kaufmann S.Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, captothecin and the other cytotoxic anticancer drugs:a cautionary note[J].Cancer Res.,1989,4(9):5870-5879.
[6]Cutolo M, Bisso A, Sulli A, Felli L, Briata M, Pizzorini C, Villaggio B. Antiproliferative and antiinflammatory effect of methotrexate on cultured differentiating myeloid monocytic cells but not on synovial macrophages from patients with rheumatoid arthritis[J].Rheumatol,2000,2(7),2551-2557.
[7]Merkle CJ, Moore IM, Penton BS, Torres BJ, Cueny RK, Schaeffer RC, Montgomery DW. Methotrexate causes apoptosis in postmitotic endothelial cells [J].Biol. Res. Nurs., 2000.2(1),4-10.
[8]Bokhari M, Carnachan RJ,Cameron NR, Przyborski SA. Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge [J].Anat.,2007,21(1),567-569.
[9]王荣,贾正平,陈立仁,等.毛细管电泳对氨甲喋呤对映体拆分及人血样中对映体含量测定研究[J].分析测试学报,2005,24(2)：19-23.
[10]Itoh T, Ono K, Koido KI.et al. Stereoselectivity of the folate transporter in rabbit small intestine:studies with amethopterin enantiomers [J].Chirality,2001,19(3):164-169.
[11]Masson E,Relling MV,Synold TW, et al.Accumulation of methotremate polyglutamates in lymphoblasts is a determinant of antileukemic effects in vivo [J].Clin Inves.,1996,9(7):73-78.
[12]Longo GS,Gorlick R, Tong WP, et al.Disparate affinities of antifolates or folylpolyglutamate synthetase from human leukemia cell [J].Blood,1997,9 (4):1241-1247.
[1]傅崇东,徐惠南.手性药物的立体选择性药物动力学[J].中国临床药学杂志,1998,7(6)：309-313.
[2]黄晓龙.美国FDA关于开发立体异构体新药的政策简介[J].中国新药杂志.2000,9(9)：650-652.
[3]李高.手性药物的临床药物动力学立体选择性[J].中国医院药学杂志,1999,19(7)：429-431.
[4]Meyer MC, Guttman DE. The binding of drugs by plasma proteins [J].Journal of Pharmaceutical Sciences,1968,57(6):895-899.
[5]Kaufmann S.Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, captothecin and the other cytotoxic anticancer drugs:a cautionary note[J].Cancer Res.,1989,4 (9):5870-5879.
[6]Cutolo M, Bisso A, Sulli A, Felli L, Briata M, Pizzorini C, Villaggio B. Antiproliferative and antiinflammatory effect of methotrexate on cultured differentiating myeloid monocytic cells but not on synovial macrophages from patients with rheumatoid arthritis[J].Rheumatol,2000,2(7),2551-2557.
[7]Merkle CJ, Moore IM, Penton BS,Torres BJ,Cueny RK, Schaeffer RC, Montgomery DW. Methotrexate causes apoptosis in postmitotic endothelial cells [J].Biol. Res. Nurs.,2000.2(1),4-10
[8]Bokhari M, Carnachan RJ, Cameron NR, Przyborski SA. Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge [J].Anat.,2007,21(1),567-569.
[9]王荣,贾正平,陈立仁,等.毛细管电泳对氨甲喋呤对映体拆分及人血样中对映体含量测定研究[J].分析测试学报,2005,24(2)：19-23.
[1]Amato I.Looking glass chemistry[J].Science,1992,256:964-968.
[2]于平.手性化合物研究进展[J].化工进展,2002,21(9)：536-538.
[3]官家乐,高原,高鸿慈.手性药物[J].中国医院药学杂志,2001,21(7)：443-444..
[4]宫丽,卞手性药物的药理学立体选择性[J].国外医学药学分册,2007,34(2)：321-324.
[5]何凤慈,陈亮,蒋耀光.酮洛芬及其手性对映体的研究进展[J].中国药房,2004,15(4)：244-246.
[6]杜玉民,刘伟娜,白希瑞.手性药物[J].临床荟萃,2003,18(20)：6914-6916.
[7]王丹,何浪,手性与手性药物[J].成都医学院学报,2006,1(2)：155-147.
[8]陈亮,何凤慈.光学活性药物的药动学和药效学差异[J].中国新药与临床杂志,2003,22(6)：377-380.
[9]蒋兆健,吴笑春.手性药物的对映体选择性与临床应用[J].中国药房,2001,12(3)：162-164.
[10]李良智.史大永,刘均洪,等.手性药物的构型与药效活性[J].青岛化工学院学报,2001,22(2)：124-126.
[11]王普善,王宇梅.手性药物开发战略的再认识[J].精细与专用化学品,2004,12(10)：4-8.
[12]Caldwell J.The importance of stereochemistry in drug action and disposition.J Clin Pharmacol,1992,32:925-927.
[13]Hutt A,Tan SC.Drug chirality and its clinical significance.Drugs,1996,52(5):1-4.
[141曾苏,沈向忠,刘志强.手性药物的药理及其体内对映体的HPLC测定.浙江医科大学学报.1994,23(5)：234-236.
[15]王似菊,沈向忠,曾苏.心血管药物的立体选择性活性及临床意义.中国医院药学杂志,1995,15(6)：284-286.
[16]Peng SX, Henson C,WilsonLJ.Sinultancous determination of enantioselective plasma protein binding of aminohydantoins by ultrafiltration and chiral hiph-performance liquid chromatography[J].Chromatogr B Biomed Sci Appl.1999;73:31-37.
[17]Menard C, Lamiable D, Vistelle R, et al. Stereoselective Biotransfomation of cicletamine in cultured rat and human hepatocytes[J].Pharmacol Res,2004;42(1):87-92.
[18]Drayer DE.Problems in therapeutic drug monitoring:the dilemma of enantiomeric drugs in man.The Drug Monitoring,1988,10(1):1-5.
[19]Zeng S,Zhang L,Liu ZQ,et al.Stereoselective metabolism of ofloxacin in human.1995, 9(1):87-90.
[20]Jamali F,Mehvar R,Pasutto.Enantioselective aspects of drug action and disposition: therapeuticpitfalls.J Pharm Sci,1989,78(9):695-699.
[21]Lee EJD,Williams KM.Chirality clinical pharmacokinetic and pharmacodynamic consideration.Clin Pharmacokinet,1990,18(2):339-343..
[22]段立华.多沙唑嗪对映体对大鼠离体心肌和豚鼠离体气管平滑肌的作用[D].河北医科大学,2007,9-14.
[23]毛小琴,邱宗荫,邱峰,等.普萘洛尔对映异构体诱导HUVEC细胞的蛋白质表达谱差异.中国生物化学与分子生物学报,2008,24(2)：165-171.