| Objective To study the difference in expression of RFC,GST-π,DHFRmRNA between human osteosarcoma U2-OS cell line and the MTX-resisitant variants U2-OS/R1-R3,and to investigate the significance in MTX resistance for human osteosarcoma.Methods Three resistant MTX human osteosarcoma cell lines were established by pulse exposure parental cell line(U2-OS) in gradually increased dose of MTX.The sensitivity of resistance for the U2-OSand U2-OS/R1-R3 to MTX,DDP,ADM and IFO were measured by MTT,the IC50 and RI were testd;The morphology of the U2-OSand U2-OS/R1-R3 were observed by inverted microscope;The proliferation ability of cell lines was measured by growth curve.The cell stage were analyzed by flow cytometry; The expression of RFC,GST-π,DHFRmRNA were assayed by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR).Results Three MTX-resistant variants(U2-OS/R1-R3) were successfully established;we fond that proliferation ability of resistant cells decreased significantly by observeding cell growth cure;Cell stage distribution of resistant cells has changed by flow cytometry;The resistant index of U2-OS/R1-R3 to MTX is 49.16,60.13,63.3 times than U2-OS by MTT;The RFC mRNA expression of U2-OS/R1-R3 is 0.7139,0.6258,0.4908 times than U2-OS(p＜0.05),The DHFR mRNA expression of U2-OS/R1-R3 is5.3145,12.8937,30.6501times than U2-OS (p＜0.05),The GST-πmRNA expression of U2-OS/R1-R3 is 1.9081,3.5420,4.8319 times than U2-OS(p＜0.05).Conclusion The results of the FQ-PCR revealed that the MTX resistance was associated with the decreased expression of the RFC mRNA and increased expression of DHFR mRNA and GST-πmRNA.We investigated the MTX resistant mechanism of human osteosarcoma cell line at a gene level.The decreased expression of RFC mRNA and the increased expression of DHFR mRNA and GST-πmRNA participate in the MTX resistance in human osteosarcoma cell lines U-2 OS. This provide the evidence for exploring the MTX resistance mechanism in clinical osteosarcoma patients,and helps to screen the patients who were insensitive to MTX chemotherapy. |
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