| Objective:To observe the effect of the telomerase inhibitor 3'-azido-2'3'-dideoxythymidine (AZT) on the rat glioma cell line (C6 cells) proliferation inhibition and induction of apoptosis, and its impact on the cell cycle. Preliminary understanding the effect of AZT on malignant glioma cell proliferation, apoptosis and cell cycle after inhibition of telomerase activity, to provide an objective test basis for the feasibility of telomerase as targets for malignant glioma to chemotherapy of targeted therapy.Methods: Disposable suction of culture medium after 12h, C6 cells were randomly divided into six groups, each hole by adding different concentrations of AZT and the culture medium, so that the final size of each hole was 2ml. using AZT treating Experimental group 1 (100μmol / LAZT), experimental group 2 (150μmol / LAZT), experimental group 3 (200μmol / LAZT), experimental group 4 (250μmol / LAZT), experimental group 5 (300μmol / LAZT) for 12h,24h, 48h, at the same time, using the same volume of DMEM culture medium and DMSO as alternative to replace AZT as control group, each concentration cultured 5 duplication hole. taking the following tests From experimental groups and control group cells: Evaluation the impact of AZT on C6 cell proliferation activity and growth rate with cell morphologyobservation; detected the survival rate of C6 cells with MTT assay, calculated the best time and concentration on the effect of AZT; detected the impact of different concentrations of AZT at different times on the apoptosis and cell cycle of C6 cells by flow cytometry (FCM) and TdT-mediated The dUTP nick end labeling (TUNEL) assay .Results: 1.Observed the cell morphology after AZT treatment, observed AZT treatment C6 glioma cells with inverted microscope ,we found that with the time and concentration increase of the drug , C6 glioma cells began to float round, diopter lower, cells connected loosely, the density was sparse, the proliferation was slow down. 2. Detected the effect of different concentrations of AZT at different time points on the proliferation and inhibition of C6 glioma cell with MTT assay, different concentrations of telomerase inhibitors AZT used on C6 glioma cells, with the inhibitor concentration increased, the role of time extension, the survival rate of tumor cells gradually reduced. Compared with the control group, 100μmol / L of AZT in the 12h ,the proliferation of C6 glioma cells have significantly inhibited (p <0.05), its inhibitory rate was 7.2%, when AZT concentration of 250μmol / L and 300μmol / L at 48h, its proliferation of C6 glioma cells significantly greater inhibition (p <0.01), their inhibitory rate reached 48.7 percent and 56.8 percent. Calculated IC50 value was approximate 269μmol / L. The experimental results show that, with the increase in concentration of AZT and the action time, the inhibition of proliferation of C6 glioma cells gradually increased in a time - dependent manner. Through the measured OD value,we calculated the best time of AZT acting on C6 glioma cells is 48h, the best concentration is 269μmol / L.3.Detected the impact of AZT on the apoptosis and cell cycle of C6 cells by flow cytometry (FCM), flow cytometry cell analysis showed that, when AZT was 100μmol / L, 48h can occur the peak of apoptosis, cell apoptosis rate was (11.63±1.115)%, cells increased on G0/G1 phase, cells increased slightly on S phase and G2 / M phase . When AZT was 300μmol / L, occoured obvious apoptotic peak at 48h, apoptosis rate was (45.17±2.566)%, compared with the control group, apoptosis rate on G0/G1 phase cells was descended from (77.23±0.702)% to (46.97±0.862)%,which was descended from (7.77±1.06)% to (33.07±1.121)% on S phase cells. Considered an effective telomerase inhibitor AZT inhibits telomerase activity, AZT may be involved in some of the cell cycle and apoptosis regulation.4.Detected the impact of AZT (IC50) on apoptosis of C6 glioma cell at 48h by TUNEL assay, the results showed that C6 glioma cells actived by AZT at the concentration of 250μmol / L after 48h. the positive rate of apoptotic cells was 27%, positive rate of necrotic cells was only 1.4%. The control group positive rate of apoptotic cells was only 5.4%, positive rate of necrotic cells was 0.9%。Conclusion:1. In vitro experiments. AZT inhibited the proliferation and growth of C6 glioma cells and its inhibition with AZT was the time - dose-dependent manner.2. Flow cytometry showed that:after AZT effectted on C6 glioma cells, the change of cell cycle is mainly expressed in increasing of S phase cells and accompanied by an increase in apoptotic cells, suggesting that the impact of AZT on C6 glioma cells may be acting on in S phase, affected their DNA synthesis and involved in apoptosis.3. TUNEL assay results showed that: In vitro, the impact of AZT on C6 glioma cells may be apoptosis in the main. The specific mechanism remains to be further studied.4. The results of this test provide the experimental basis for the AZT as a target on telomerase for the treatment of brain glioma .
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