5-氮杂胞嘧啶核苷诱导小鼠骨髓间充质干细胞凋亡  

5-azacytidine induces apoptosis of bone marrow mesenchymal stem cells in mouse

作　　者：武俊芳[1] 吴艳芳[2] 李晓鹏[4] 张芬熙[3,5] 林俊堂 高利洁[1] 崔柳苏[1] 

机构地区：[1]河南新乡,新乡医学院形态学实验室,453003 [2]河南新乡,新乡医学院形态学解剖实验室,453003 [3]干细胞与生物治疗研究中心 [4]新乡医学院第三附属医院眼科 [5]新乡医学院三全学院解剖教研室

出　　处：《中华实用儿科临床杂志》2014年第17期1337-1339,共3页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金（31000475）

摘　　要：目的研究5-氮杂胞嘧啶核苷（5-AZA）对小鼠骨髓间充质干细胞（BMSCs）凋亡的影响。方法从小鼠股骨和胫骨骨髓中分离BMSCs；利用免疫荧光染色检测间充质干细胞特异性标记分子CD44和CD90在BMSCs上的表达；应用0、10和20μmol／L的5-AZA体外诱导培养BMSCs48h，应用4′，6-二眯基-2-苯基吲哚（DAPI）染色和荧光标记的半胱天冬酶抑制剂（FLICA）凋亡试剂盒检测5-AZA处理后细胞的凋亡率；应用Westernblot检测凋亡相关蛋白AnnexinV和Caspase-3在处理后的细胞表达水平。结果实验中，BMSCs阳性表达间充质干细胞特异性标记蛋白CD44和CD90。DAPI染色和Caspase-3染色均显示10和20μmol／L5-AZA可增加BMSC的凋亡率；对照组、10μmol／L组5-AZA和20μmol／L5-AZA组DAPI染色阳性凋亡率分别为（21．086±2．601）％、（34．467±3．724）％和（46．512±3．864）％，Caspase-3染色阳性凋亡率分别为（5．354±0．735）％、（15．4624-2．385）％和（28．190±4．190）％，组间比较差异均有统计学意义（P均＜0．01）。Westernblot检测显示AnnexinV和Caspase-3在5-AZA处理后表达均显著增高；对照组、10μmok／L组5-AZA和20μmol／L5-AZA组AnnexinV相对表达水平分别为（26．612±2．184）％、（42．873±4．313）％和（50．056±4．457）％，Caspase-3的相对表达水平分别为（19．231±2．683）％、（38．618±5．385）％和（91．235±7．116）％，组间比较差异均有统计学意义（P均＜0．01）。结论常规剂量的5-AZA可诱导BMSCs凋亡。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） stai- ning ; 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 Ixmol/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 apop- tosis-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 Ixmol/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 C aspase-3 expression was （ 19.231 ± 2. 683 ） % , （ 38.618 ±5. 385 ） % and（91. 235± 7.116）% in the controls, 10 and 20 p.mol/L 5-AZA groups, and there were significant signifirences 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.

关 键 词：5-氮杂胞嘧啶核苷 骨髓间充质干细胞 细胞凋亡 CASPASE-3表达 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]