| Background: Methotrexate (MTX) is a significant drug precursors of folic acid-like anticancer, with two kinds of enantiomers: L-(+)-MTX and D-(-)-MTX,which was widely used in treatment of lymphoma, acute leukemia and lung cancer and other tumor.As one of the key-enzymes in folic acid metabolic pathway, Folylpolyglutamate synthetase (FPGS) content and activity directly associated with the effects of MTX. At present, many researches concentrate themselves on FPGS study, but the quantitative detection of FPGS was rarely reported.Objective: Firstly, to design a detection method of FPGS, secondly optimize the experiment conditions, thirdly evaluate the specificity, separation, and sensitivity of the designed method,lastly to analyse the test results by statistical software. Observing the change of FPGS contents in the drug-resistant cell strains that evolved from A549 after being induced by different methotrexate (MTX) enantiomer with this method, to provide new research platform for future study of tumor drug resistance mechanismsMethods: First, 25μmol / L L-(+)-MTX drug resistant cell strains and 25μmol / L D-(-)-MTX drug resistant cell strains were established by using large doses of incremental drug method, and sensitive cell strains as a control in FPGS expression. All the three cell strains were repeatedly freezed and thawed by using liquid Nitrogen, completely destroy cells, collect cell protein extract of the supernatantby the way the three strains wet weight about 100 mg,;Analysis the extract with the SDS-polyacrylamide electrophoresis(SDS-PAGE) and Western blot(WB);Use fluorescein isothiocyanate (fluorescein isothiocynate, FITC) derivatives marked FPGS antibody with dialysis method,then reaction with the extract in room temperature and dark room,for 30 min .The non-competitiveimmune reaction have strong specific;after reaction,using immune capillary electrophoresis - laser-induced fluorescence detection technology (CEIA-LIF) isolate marked FPGS antibody and FPGS antigen-antibody mixture, CEIA-LIF ,include capillary micellar electrokinetic chromatography (MEKC) detection technology and laser-induced fluorescence detector (LIF) technology, can detect the FPGS expression in different MTX enantiomers affects the lung cancer A549 cell strains.Results: FPGS molecular weight is about 66 000D with confirmation by SDS-PAGE experiments ,and there was no other band came forth which was confirmed by WB experiments. After the gel imaging system preliminary quantitive analysis, FPGS expressions of 25 mol / L-(+)-MTX drug resistant cell strains and 25 mol / L D-(-)-MTX drug resistant cell strains are 52.14%and 60.06% compare with sensitive cell strains respectively.Useing CEIA-LIF test method, the process of separation takes 7.1 min in separation of marked FPGS antibody, at the same time it takes 8.9 min in separation of immune complexes. In both process it can be separated and detected in no more than 10 mins,and the resolution threshold (R) is 4.5.With the method of CEIA-LIF detection cells FPGS expression, the FPGS expressions of 25 mol / L-(+)-MTX drug resistant cell strains and of 25 mol / L D-(-)-MTX drug resistant cell strains are 46.59%and 48.36%respectively,compareed with sensitive cell strains. These results accompanied with the quantitative results of classic WB preliminary were statistical analysesed by the statistical software SPSS (Version:13.0) Weld pattern,P>0.05, demonstrated there no significant difference.Conclusions: CELA-LIF is of generally fast efficient in detecting FPGS, the determination time is short,and similar to WB method, with high specificity and high accuracy;With fluorescent markered, MECC separation technology and LIF detection system useing in this new method, the detection specificity, sensitivity greatly improved. The expression consents of FPGS in L-(+)-MTX and D-(-)-MTX drug resistant cell strains is damaged and decreased obviously. |
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