Research On Establishment Of Drug-resistance Cell Line Of Human Glioma Induced By TMZ And Its Reversion Of Drug-resistance To TMZ

Part one:Establishment of Drug-resistance Cell Line of Human Glioma Induced by TMZObjective Giomas aren't cured completely with surgery ,temozolomide as a newly methylating agent possesses effective clinical activity against gliomas. However, the recent clinical trials show clinical resistance to temozolomide(TMZ) limits its efficacy in the therapy of gliomas, and becomes a major problem to gliomas chemotherapy.Therefore ,the establishment of drug-resistance human glioma cell line to TMZ has significant value for the study of revision of gliomas drug-resistance and improvement of chemotherapeutic approaches .Methods First,the basic expression of MGMT mRNA for U251 was assayed by RT-PCR method,Chemotherapeutic resistance in U251 human glioma cells was caused by the stepwise revulsion with TMZ. The resistance index and the cell colony were assayed; We assayed the cytotoxicity of TMZ to U251/TR by MTT method,calculated the index of durg-resistance to U251/TR by regression equation and calculated durg-resistance to U251/TR.Finally, the expression of MGMT mRNA for U251 was assayed by RT-PCR method; Results was analyzed between U251/TR and U251.Result No basic expression of MGMT mRNA for U251 was assayed by RT-PCR method; U251/TR was established by the stepwise revulsion with TMZ;The cell cloning experiment demonstrated that the colony-foming efficency of U251/TR-6 cells induced stepwisely with TMZ was 4~11 folds than that of U251 cells without inducement in cell culture with 20.61～164.86mM TMZ. U251/TR showed about 4 fold resisitance to TMZ assayed by MTT method. Meanwhile, RT-PCR result showed that U251/TR cells expressed up-regulated MGMT mRNA which could be amplificated out for a 571-bp specific fragment.Conclusion A constant drug-resistace cell line U251/TR induced by TMZ was established successfully in 6-month culture,meanwhile,increasing the concentration of TMZ stepwisely was the important link; MGMT played a very important role in the drug-reisistance acquired by U251 cell to TMZ.Part two:Research on reversion of drug-resistance in human glioma U251/TR by O~6-BG of the inhibitor of MGMTObjective TMZ is the one of the most effective chemotheraptic drug ,however, drug-resistance limited the its clinical result. Research show MGMT to DNA protein for repaired was the main molecular drug-resistance mechanism on TMZ et al. The objective was to detect cytotoxic effective of TMZ combined with O~6-BG to Mer-and Mer+ giomas pretreated by O~6-BG,and explore the path of TMZ to giomas chemotherapy.Methods We used the human glioma cells induced by TMZ and U251 cell to make the experment . Our experiment was divided into four groups; 1) control group ;2) O~6-BG group 3) TMZ group 4) O~6-BG+TMZ group ; A standard MTT assay was used to evaluate the ability of O~6-BG to modulate the cytotoxicity of TMZ .Result 3.1tiems of elevation of sensitivity in U251/TR to TMZ was observed ,while has little elevation of sensitivity in U251.10μM O~6-BG could obviously deplete MGMT activity of U251/TR cell and complete consumption was observed when more than 50μMof O~6-BG was used. O~6-BG has a little toxicity and the survival rate of cells was more than 95% in 50μM.Conclusion O~6-BG had the less cytotoxicity , which hasn't the significant influence on parental cell and drug-resistance cell line; By delepting MGMT activity , O~6-BG could reverse TMZ durg-resistance of U251/TR cells to TMZ, which suggested that O~6-BG may be a useful chemotherapeutical sensitizer combined with TMZ to gliomas.