中文摘要：

目的诱导并建立耐氨甲蝶呤对映体的A549细胞株并观察耐药细胞系（L-（＋）-MTX／A549、D-（-）-MTX／A549）的生物学特性。方法以MTX对映体为诱导剂，采用浓度递增结合低剂量持续诱导方法诱导A549细胞株，建立MTX不同对映体耐药细胞系；倒置相差显微镜观察细胞形态变化；MTT法绘制细胞生长曲线；MTT法检测耐药细胞株的耐药指数；流式细胞仪检测细胞周期和细胞的分裂增殖能力。结果L-（＋）MTX／A549、D-（-）MTX／A549耐药指数分别为6．0的和20．2。倒置相差显微镜观察细胞形态发生了改变；细胞生长曲线显示D-（-）-MTX／A549的增殖略慢于亲本细胞，而L-（＋）-MTX／A549的增殖最慢；流式细胞仪检测细胞周期结果显示L-（＋）MTX／A549、D-（-）-MTX／A549耐药细胞株S期细胞数量减少（P〈0．05），G0／G1期细胞增多（P〈0．05）；CFSE检测A549、L-（＋）-MTX／A549、D-（-）-MTX／A549的MFI分别为（6．08±0．55）、（7．72±0．30）、（6．90±0．18）。两对映体细胞株间有明显手性差异。结论本研究建立了MTX两种对映体耐药细胞株，为进一步研究其耐药机制提供了一种实验模型。

英文摘要：

Objective To establish methotrexate（MTX） enantiomers resistant human NSCCL A549 cell line （ L（ ＋ ）-MTX/A549,D（ - ）-MTX/A549） derived from the A549 cell line and observe its biological characters. Methods A549 cells were exposed to intermittently and progressively increasing doses of MTX enantiomers. Two MTX enantiomers （L and D） resistant human NSCLC A549 cell lines were established. The morphology was observed by inverted phase contrast microscope, the sensitivity of the L- （ ＋ ）-MTX/A549, D-（ - ） MTX/A549 cell lines to MTX enantiomers was measured by cytotoxicity test. The cell growth curve was determined by MTT assay, the change of CFSE fluorescence intensity and cell cycle distribution were analyzed by flow cytometry assay. Results The L（ ＋ ）-MTX/A549 resistance index was 6. 0 and D-（ - ）-MTX/A549 resistance index was 20. 2. Inverted phase contrast microscope showed morphological changes of L-（ ＋ ）-MTX/A549 and D-（ - ）-MTX/A549. Cell growth curve demonstrated that the proliferation abilities of L-（ ＋ ） MTX/A549 was decreased significantly, while the cell proliferation rate of D-（ - ）-MTX/A549 was similar to that of A549. The flow cytometry results showed L-（ ＋ ）-MTX/A549 and D-（ - ） MTX/A549 cells at S phase were reduced （P〈0. 05） , with an increased at G0/G1 phase （P〈0. 05）, the CFSE mean fluorescence intensity of A549 parent cells, L（ ＋ ）- MTX/A549 and D-（ - ）-MTX/A549 were （6. 08 ±0. 55）, （7. 72 ±0. 30）, （6. 90 ± 0. 18）. Two enantiomers resistance cell lines have a clear difference in Chirality. Conclusion We established two MTX enantiomers resistant human A549 cell lines and observed their different biological characteristics. It provides a new experimental model for further studying the mechanism of MTX enantiomers resistance.