ABSTRACT
Objective To investigate the effect of 5-azacytidine(5-AZA) on apoptosis of bone marrow mesenchymal stem cells(BMSCs).Methods BMSCs were isolated from bone marrow of mouse tibia and femur; the expression of MSC specific markers CD44 and CD90 in BMSCs was measured by immunofluorescence staining;BMSCs were cultured in vitro in the medium supplemented with 0,10 and 20 μmol/L 5-AZA for 48 hours.Cell apoptosis was measured with fluorescent labeled inhibitor of caspases (FLICA) apoptosis kit and 4',6-diamidino-2-phenylindole (DAPI) staining ;the expression of apoptosis-related proteins Annexin V and Caspase-3 in the treated BMSCs was detected by Western blot.Results In this study,BMSCs positively expressed MSC specific markers CD44 and CD90.DAPI staining and Caspase-3 staining both showed that 10 and 20 μmol/L 5-AZA markedly increased apoptotic rate of BMSCs;the apoptosis-positive rate in DAPI staining was (21.086 ± 2.601) ％,(34.467 ± 3.724) ％ and (46.512 ± 3.864) ％,the apoptosis-positive rate in Caspase-3 staining was (5.354 ± 0.735)％,(15.462 ± 2.385)％ and (28.190 ± 4.190)％ in the controls,10 and 20 μmol/L 5-AZA groups,and there were significant differences among the control group and 5-AZA treated groups(all P ＜0.01).Western blot assay showed that Annexin V and Caspase-3 were both markedly upregulated in 5-AZA treated cells;the relative level of Annexin V expression was(26.612 ±2.184)％,(42.873 ±4.313)％ and (50.056 ± 4.457) ％,the relative level of Caspase-3 expression was (19.231 ± 2.683) ％,(38.618 ± 5.385) ％ and(91.235 ± 7.116)％ in the controls,10 and 20 μmol/L 5-AZA groups,and there were significant differences among the control group and 5-AZA treated groups (all P ＜ 0.01).Conclusion The commonly used doses of 5-AZA can induce apoptosis of BMSCs.