Effect Of Hypoxic Microenvironment On Drug-resistant Glioma Strain MGMTE Effect Of Protein Expression

Objectives:The most common intracranial tumors are gliomas.Surgery,radiotherapy and chemotherapeutic therapy with temozolamide TMZ have become the first line of clinical treatment.The anticancer effect of TMZ is mainly mediated by 06-meG.However,in the DNA repair system of tumor cells,MGMT can repair the chemotherapeutic effect mediated by 06-meG,resulting in a decrease in the efficacy of TMZ.Inherent or acquired drug resistance limits the clinical use of TMZ.In particular,with the increase in the number and frequency of drug use,the drug resistance gradually changed from sensitive tumor to drug resistant tumor.Department.With the first evidence of hypoxia in tumor cells and the induction of changes in their biological behavior,malignant transformation of the tumor is accelerated,such as inhibition of cell proliferation,enhancement of drug resistance gene expression,selective anti-apoptosis to promote tumor invasion and metastasis.Down-regulate DNA repair gene expression and increase gene instability.Hypoxic microenvironment significantly reduced the efficacy of radiotherapy,chemotherapy and surgery.So is the expression of MGMT affected by hypoxia?does the sensitivity of MGMT change with it?The mechanism of drug resistance is needed to be explored in this experiment.In this study,three kinds of HAC cells,T98G?U251?HAC,were used.The chemosensitivity of glioma cell lines was studied under different hypoxic concentrations and different concentrations of TMZ.The drug-resistant glioma cell lines were established and the MGMT proteins related to chemotherapeutic drug resistance were screened by the new glial cell lines.It was confirmed that the protein expression was analyzed at the same time.Methods:A gradient hypoxia microenvironment was established by using a three-air incubator to emulate the hypoxic microenvironment required by gliomas.The three kinds of HAC cells of T98G U251 HAC were divided into two groups:experimental group and control group under the condition of 0.5 and 21(normoxic),the former three conditions were the experimental group and the normoxic condition was the control group.Culture with DMEM88%10s 0.5%P/S 0.5HEPES),record the changes of each stage,simulate the procedure of clinical use of TMZ,and adopt the constant drug concentration of 0.2g 0.1L 0.05U 0.0250.01250.00 625 g/L,in the form of periodic action,for 79 days);in the logarithmic growth period,the cells were cultured to resume growth,repeat the drug addition process,and induce the drug resistance until the drug resistance was stable,according to the color change of the culture medium.The rate and density of cell growth were replaced in time,and the drug-resistant strains were screened out.The cell growth curve,cell growth inhibition rate and the changes of cell morphology were observed by MTT experiment.To provide a basis for the determination of drug concentration and the selection of detection time window in vitro drug sensitivity test,the established drug resistant cell line was named as T98G/TMZ,U251/TMZ,HAC/TMZ.Western blotting was used to detect the expression and content of MGMT under different oxygen concentration.Results:T98G/TMZ.U251/TMZ,TMZ concentration using constant concentration to establish HAC/TMZ resistant cell lines,periodic treatment,after treatment process is repeated 6 times in total,which lasted 6 months,successfully established a stable resistance to T98G/TMZ.TMZ U251/TMZ,resistance index of HAC/TMZ three cells were:T98G/TMZ(RF)=10.84,U251/TMZ(RF)=8.41,HAC/TMZ(RF)=11.11,that is the degree of tolerance is about 11 times,8.5-fold,11-fold.The three new drug-resistant strains were cultured under 0.5%oxygen concentration of 1%oxygen concentration and 3%oxygen concentration,respectively.When the concentration of TMZ was taken as the observation point on the seventh day,the cell body was mainly formed,the nucleus became larger and the heterogeneity increased.The more significant changes were the morphological changes of many elongated pseudopodia cells in various directions.The results of MTT test showed that there were differences among the three kinds of cells under different oxygen concentration,especially the inhibition rate decreased when the oxygen concentration was 1%.Survival rate increased.MG detected by Western blotting There was significant difference in the content of MT protein between T98G and U251 cells.There was no significant difference in the expression of MGMT protein between T98G and U251 cells under 0.5%oxygen concentration.Conclusion(s):T98G/TMZ,U251/TMZ,TMZ concentration using constant concentration to establish HAC/TMZ resistant cell lines,periodic treatment,after treatment process is repeated 6 times in total,which lasted 6 months,successfully established a stable resistance to T98G/TMZ,TMZ U251/TMZ,resistance index of HAC/TMZ three cells were:T98G/TMZ(RF)=10.84,U251/TMZ(RF)-8.41.HAC/TMZ(RF)=11,11,that is the degree of tolerance is about 11 times,8.5-fold,11-fold.The three new drug-resistant strains were cultured under 0.5%oxygen concentration of 1%oxygen concentration and 3%oxygen concentration,respectively.When the concentration of TMZ was taken as the observation point on the seventh day,the cell body was mainly formed,the nucleus became larger and the heterogeneity increased.The more significant changes were the morphological changes of many elongated pseudopodia cells in various directions.The results of MTT test showed that there were differences among the three kinds of cells under different oxygen concentration,especially the inhibition rate decreased when the oxygen concentration was 1%.Survival rate increased.MG detected by Western blotting There was significant difference in the content of MT protein between T98G and U251 cells.There was no significant difference in the expression of MGMT protein between T98G and U251 cells under 0.5%oxygen concentration.There was no difference in drug resistance of the same drug resistant strain under different hypoxia conditions,that is,tumor heterogeneity existed,and MGMT might have synergistic effect with other drug resistance mechanisms under moderate hypoxia oxygen concentration(1%oxygen concentration),resulting in drug resistance.