CAJ | 학술논문

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20 μmol/L的5-AZA体外诱导培养BMSCs 48 h,应用4',6-二脒基-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Western blot检测凋亡相关蛋白Annexin V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20 μmol/L 5-AZA可增加BMSC的凋亡率;对照组、10 μmol/L组5-AZA和20 μmol/L 5-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)％、(15.462±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Annexin V和Caspase-3在5-AZA处理后表达均显著增高;对照组、10 μmol/L组5-AZA和20μmol/L 5-AZA组Annexin V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.
목적 연구5-담잡포밀정핵감(5-AZA)대소서골수간충질간세포(BMSCs)조망적영향.방법 종소서고골화경골골수중분리BMSCs;이용면역형광염색검측간충질간세포특이성표기분자CD44화CD90재BMSCs상적표체;응용0、10화20 μmol/L적5-AZA체외유도배양BMSCs 48 h,응용4',6-이미기-2-분기신타(DAPI)염색화형광표기적반광천동매억제제(FLICA)조망시제합검측5-AZA처리후세포적조망솔;응용Western blot검측조망상관단백Annexin V화Caspase-3재처리후적세포표체수평.결과 실험중,BMSCs양성표체간충질간세포특이성표기단백CD44화CD90.DAPI염색화Caspase-3염색균현시10화20 μmol/L 5-AZA가증가BMSC적조망솔;대조조、10 μmol/L조5-AZA화20 μmol/L 5-AZA조DAPI염색양성조망솔분별위(21.086±2.601)％、(34.467 ±3.724)％화(46.512±3.864)％,Caspase-3염색양성조망솔분별위(5.354±0.735)％、(15.462±2.385)％화(28.190 ±4.190)％,조간비교차이균유통계학의의(P균＜0.01).Westernblot검측현시Annexin V화Caspase-3재5-AZA처리후표체균현저증고;대조조、10 μmol/L조5-AZA화20μmol/L 5-AZA조Annexin V상대표체수평분별위(26.612±2.184)％、(42.873 ±4.313)％화(50.056±4.457)％,Caspase-3적상대표체수평분별위(19.231 ±2.683)％、(38.618 ±5.385)％화(91.235 ±7.116)％,조간비교차이균유통계학의의(P균＜0.01).결론 상규제량적5-AZA가유도BMSCs조망.
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.