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The Expressions of Dihydrofolate Reductase Genes and Proteins in Methotrexate Enantiomers- Resistant A549 Cell Lines of Human NSCLC

Author: LiDaoJing
Tutor: ShenZuoJun
School: Anhui Medical University,
Course: Clinical Laboratory Science
Keywords: Dihydrofolate reductase methotrexate enantiomer resistance A549 cell line Methotrexate Enantiomer Purify
CLC: R96
Type: Master's thesis
Year: 2011
Downloads: 5
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Abstract


Objective:To study the relationship between the resistance to methotrexate (MTX) enantiomers and the expressions of Dihydrofolate reductase(DHFR)genes.Methods:A549 cells were exposed to intermittently and progressively increasing dose of each MTX enantiomer. The study detected the expressions of DHFR genes by real - time fluorescence quantitative polymerase chain reaction ( FQ– PCR ).Results:The resistant index had difference between L-(+)-MTX and D-(-)-MTX enantiomer resistant cell lines. D-(-)-MTX resistance cell lines have higher resistant index than L-(+)-MTX resistant cell lines. The levels of DHFR gene expression has difference in 15μmol/L MTX between resistant cell lines of L-(+)- and D-(-)-MTX, with lower level in D-(-)-MTX resistant cell line.In addition, quantitation of DHFR mRNA revealed a remarkable increase of DHFR gene expression in 35μmol/L and 45μmol/L MTX resistant cell lines with higher level in D-(-)-MTX resistant cell line. The results of the FQ - PCR revealed that the MTX resistance was associated with overexpression of DHFR genes. Conclusions:The expressions of DHFR genes was inhibited after the cell lines induced by 15μmol/L MTX enantoimers with lower level in D-(-)-MTX resistant cell line. The expression of DHFR genes had a significant difference in chirality. The results revealed that the expressions of DHFR genes may be examined in MTX treatment phase which could help to prognosticate the cell’s resistant status and D-MTX is not be regarded just as a contaminant in the drug MTX. MTX with single enantiomer (L-MTX) should be selected clinically so as to decrease the side effects of D-MTX. Objective: To study the relationship between the resistance to MTX enantiomers and the expressions of DHFR proteins,and to purify dihydrofolate reductase from A549 parent cell line of Human NSCLC.Methods: The study detected the expressions of DHFR proteins by Western blot analysis. Collect culture cell and purify dihydrofolate reductase by preparation of cell extract→PH5.0 supernatant fraction→ammonium sulfate fractionation→ultrafiltration→DEAE-sepharose Fast Flow ion exchange chromatography→ultrafiltration. SDS-PAGE was performed to determine the molecular weight of dihydrofolate reductase. The final purification protein was confirmed by Western blot analysis.Protein concentration was measured by using the Coomassie brilliant blue method .The activity of dihydrofolate reductase was assayed using a spectrophotometer.Results: The expressions of DHFR proteins has difference in 15μmol/L MTX between resistant cell lines of L-(+)- and D-(-)-MTX, with lower level in D-(-)-MTX resistant cell line. The high expression levels of DHFR proteins in 35μmol/L and 45μmol/L MTX resistant cell lines with higher level in D-(-)-MTX resistant cell line were detected(P<0.05). Moreover, the study succeed in extraction and purification of dihydrofolate reductase in the A549 parent cell line. Conclusions: DHFR could play important role in the resistance to MTX enantiomers. Besides, The results show that the comparatively pure dihydrofolate reductase have been obtained through these procedures. So, it will lay the foundation for further study of MTX chiral drug-resistant mechanisms.

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CLC: > Medicine, health > Pharmacy > Pharmacology
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