| Purpose:To discuss the drug-resistance mechanism of cerebral glioma basing on the concept of brain tumor stem cell. To discuss the alteration of the expression of stem cell gene and drug resistant gene in the process of drug resistance formation, by generating drug resistant cell lines with ACNU induction, and to discover the relationship between them, so as to provide guidance for individualized treatment.Methods:The astrocytoma cell strain was provided by Peking university health science center. Those cells were cultured in medium containing ACNU with concentration of100ug/ml for5generations, then the drug was withdrawn and the drug resistant cell strain was generated. The difference of ACNU resistance in tumor cells before and after ACNU induction was compared by CCK-8method quantitively. The alteration of CD133positive cells proportion before and after drug induction were analysed by flow cytometry. The expression of stem cell related genes CD133, Nestin, OCT4and Sox2, and drug resistance genes MGMT and MDR1were tested by quantitative real time PCR. The methylation level in the promoter area of MGMT gene was tested by methylation specific high resolution heat-melting method.Results:After ACNU induction, the ACNU tolerance of glioma cells were significantly increased. The proportion of CD133positive cells in drug resistant cell group and control group were0.49%and9.74%respectively, the number of CD133+cells were significantly decreased. The expression of stem cell related genes in drug resistant cell were down regulated compared with control group. Especially, the expression of OCT4gene, one of the stem cell genes, had a remarkably down regulation. The expression of MGMT gene had a56.6times up regulation, which was statistically significant. The expression of MDR1gene in both group were very low. The methylation level of MGMT gene promoter had no significant difference between drug resistant group and control group.Conclusion:The drug resistant cell strain could be generated by short time induction of large dose of ankylating drug ACNU. In drug resistant cell strain generated by this means, the proportion of brain tumor stem cells remarkably dropped and the expression of other stem cell related gene down regulated in various degrees. Meanwhile, the expression of drug resistant gene MGMT was significantly increased, however, the methylation level of MGMT gene promoter and the amount of its gene products were not consistent. The mechanism of induced drug resistance in this experiment is mainly from high expression of MGMT gene. |
PDF Full Text Request