| Objective:This experiment using valproate(valproic acid, VPA) and rapamycin treat multiple myeloma(multiple myeloma, MM) cell line RPMI8226 and U266 cells in vitro to observe apoptosis level changes,and to explore the relationship between cell apoptosis and autophagy in multiple myeloma, and provide experimental data for clinical treatment of MM by VPA.Methods:The MM cell line RPMI8226,U266 cells were cultured in vitro, experimental groups: control group, 2,4,8mmol/L VPA group, 250nmol/L rapamycin, 8mmo/L VPA+250nmol/L rapamycin combination group. Flow cytometry was used to detect apoptosis of RPMI8226 and U266 cell lines with VPA and rapamycin treated on different time, real-time quantitative PCR(Real-time PCR, RT-PCR) and western blot to detect the expression of apoptosis-related factors BAX, Bcl2, Caspase3, Caspase8 m RNA and protein.Results:1 Flow cytometry detect MM cell ApoptosisThe results show: the apoptosis of 2,4,8 mmol / L VPA teated RPMI8226 cell line 48 h were 4.33±0.37%, 17.35±0.59%, 30.11±0.76%; and apoptosis U266 cells were 5.17±0.71%,25.53±0.59%,38.33±2.11%,different concentrations of VPA treatment of multiple myeloma cell lines RPMI8226 and U266, with VPA concentrations increased RPMI8226 and U266 cells Apoptosis gradually increased,the difference was statistically significant(P<0.05). 8mml/L VPA treated RPMI8226 and U266 cells 0h, 12 h, 24 h, 48 h, RPMI8226 cell apoptosis were 1.68±0.32%, 9.93±0.47%, 15.51±0.62%, 30.11± 0.76%; the apoptosis of U266 cells were 2.27±0.27%, 13.14±2.18%, 25.76±1.83%, 38.33±2.31%, with prolonged duration of action of VPA, the apoptosis of RPMI8226 and U266 cells gradually increased, the difference was statistically significant(P<0.05). different drug treatment RPMI8226 cell apoptosis after 48h: control group: 4.43±0.30%, VPA group: 30.11± 0.76%, rapamycin: 25.36 ±0.68%, VPA+rapamycin:43.51±0.71%. different drug-treated U266 apoptosis after 48h: control group: 5.66±0.80%, VPA group: 38.33±2.11%, rapamycin: 22.72±2.61%,VPA+rapamycin:44.68±3.32%,The VPA group,rapamycin group and VPA and rapamycin combination group compared with the control group the apoptosis increased and the most is combined group.2 From the results of flow cytometry VPA could promote MM cell apoptosis, and induced apoptosis in a dose-time dependent manner, the VPA and rapamycin promote MM cell apoptosis has a synergistic effect, so the apoptosis gene and protein level how they effect,we use real-time PCR and western blot method to detect apoptosis related gene Bax, Bcl2, Caspase3, Caspase8 m RNA and protein expression,The results showed that different concentrations of(2,4,8mmol/L)VPA treated RPMI8226 and U266 cells 12 h, 24 h, 48 h, with the increased of VPA concentration and duration time, Bax, expression Caspase3, Caspase8 m RNA and protein level increased, Bcl2 m RNA expression and protein level decreased, the difference was statistically significant(P <0.05).To further analyze the impact of rapamycin with VPA alone and in combination on apoptosis gene and protein level. The results show: VPA monotherapy group and rapamycin monotherapy group compared with the control group, apoptosis related factor Bax, caspase3, caspase8 m RNA and protein expression increased, reducing the amount of bcl2 m RNA and protein expression, the difference statistically significant, and the VPA with rapamycin combined group of pro-apoptotic factor Bax, caspase3, caspase8 expression was significantly increased, significantly reduced the expression of antiapoptotic factor bcl2.Conclusion:1 VPA can promote multiple myeloma cell apoptosis in a time-dose dependent manner.2 After VPA combined rapamycin the pro-apoptotic factor Bax, caspase3, caspase8 expression increased significantly, significantly reduce the amount of expression of anti-apoptotic factor bcl2.3 VPA and rapamycin have a synergistic effect in promoting multiple myeloma RPMI8226 and U266 cell lines apoptosis... |
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