Establishment Of Methotrexate Enantiomers Resistant A549 Cell Lines Of Human NSCCL And Purification Of Dihydrofolate Reductase

Part A:Establishment of Methotrexate Enantiomers Resistant A549 Cell Lines of Human NSCCL and Its Biological CharacteristicsObjective 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 dose of MTX enantiomers.Two MTX enantiomers(L-(+)-MTX and D-(-)-MTX) resistant human NSCCL 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 MTX enantiomers resistant human A549 cell lines were established.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 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. Part B:Purification of Dihydrofolate Reductase from Methotrexate Enantiomers Resistant A549 Cell LinesObjective:To purify dihydrofolate reductase from A549 parent cell line and methotrexate enantiomers resistant A549 cell lines.Methods:Collect culture cell and purify dihydrofolate reductase by preparation of cell extract→PH5.1 supernatant fraction→ammonium sulfate fractionation→sephadex-25 chromatograph→ultracentrifugation→DEAE-sepharose Fast Flow ion exchange chromatography→ultrafiltration.SDS-PAGE was performed to determine the molecular weights of dihydrofolate reductase.Protein concentration was measured using the Coomassie brilliant blue method.the activity of dihydrofolate reductase was assayed using spectrometerically.Results:Successful extraction and purification of dihydrofolate reductase in the A549 cell line and methotrexate resistant enantiomers A549 cell lines.The dihydrofolate reductase was purify by 48 fold apparent homogeneity,with a 41.4%yield.Conclusion:The results suggest that the pure dihydrofolate reductase have been be obtained through these procedure.For further study of MTX chiral drug-resistant mechanisms to lay the foundation...