| Purpose: Trough establishing the resistant methotrexate human osteosarcoma Saos-2/mtx4.4 cell line ,study its biologcal characteristics and future the resistance of medicineMethod: A resistant methotrexate human osteosarcoma cell line (Saos-2/mtx4.4) was established by pulse exposure parental cell line (Saos-2 )in gradually increased dose of MTX . The sensitivity of resistance for the Saos-2,Saos-2/MTX2.2 and Saos-2/MTX4.4cell lines to MTX,DDP,EPI,ADM,THP and PTX were measured by MTT way assay . The inhibitory rate of tumor cells proliferation (%) equal to(1-OD value of tested group/OD value of control group)×100%. The resistant index egual to IC50(resistant cell)/ IC50 (parental cell).The morphology and ultramicro structure of the cell lines were observed by optical microscopy and transmission electronic microscopy. The proliferation ability of cell lines was measured by growth curve. The cell stage were analyzed by flow cytometry. The reverse transcription polymerase chain reaction(RT-PCR ) was utilized to evaluate the express of RFC(reduced folate carrier ,RFC)gene to Saos-2,Saos-2/MTX2.2 and Saos-2/MTX4.4 cell lines and compare gene express by semi- quantitative way. 3H-MTX inside of Saos-2 and Saos-2/MTX4.4 cell lines was measured by liquid scintillation analgzer in order to evaluate the function of RFC gene in Saos-2 and Saos-2/MTX4.4 cell lines.Result: Through induced after 27 weeks, we established different drug concentration induced cell lines(Saos-2/MTX1.1,Saos-2/MTX2.2 and Saos-2/MTX4.4) . Saos-2/MTX2.2,Saos-2/MTX4.4 cell line was utilized to resistant experimentation. Cell morphology was observed through light microscopy. The resistant cells are multangularer, and coenocyticer than parental cell. The decrease of cell villus and the increase of nucleoli in resistant cells were observed through transmission electronic microscopy. Through observeding cell growth cure , we fond that proliferation ability of resistant cells decreased significantly . Cell stage distribution of resistant cells has changed. The G0 and G1 stage percentage of resistant cell lines increased. The S stage percentage of resistant cells decreased. The resistant index of Saos-2/MTX2.2 and Saos-2/MTX4.4 cell lines to MTX is 4.87 and 12.73 times than Saos-2 cell line differently, resistant indexes of Saos-2/MTX4.4 cell lines to doxorubicin, Ifosfamide, Epirubicine, Pirarubicin and Paclitaxel were 3.94, 4.02, 4.09, 3.15 and 2.44 diffdrently (P<0.05). The cell was found to remain sensitive to cisplatin (P>0.05). RFC gene mRNA expression level of resistant cells was decreased gradually along with the concentration increasing of MTX in resistant cell lines. Through measuring 3H-MTX quantitative within cells by liquid scintillation analgze, we found that The time of reaching drug peak concentration in different cell lines is all 30 minutes, The time of reaching planus concentration is all 50 minutes, drug concentration inside of Saos-2 in planus concentration stage is 2.15 times than resistant cells.Conclusion: We established resistant drug human osteosarcoma cell line( Sao-2/MTX4.4) in order to offer experiment model for overcome tumor resistance research and research the resistant mechanism of cell in gene level. |
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