Oct-4表达下调促大鼠骨髓多能成体祖细胞向心肌分化  

Downregulation of Oct-4 expression accelerates cardiomyocyte differentiation of rat bone marrow multipotent adult progenitor cells in vitro

作　　者：谢靖芸[1] 吕铁伟[1] 朱静[2] 陈国珍[1] 孙慧超[1] 刘振国[3] 田杰[1] 

机构地区：[1]重庆医科大学附属儿童医院心脏内科 [2]儿童发育疾病研究省部共建教育部重点研究室,重庆400014 [3]美国俄亥俄州立大学医学中心心肺研究所,哥伦布43210

出　　处：《重庆医科大学学报》2011年第5期564-568,共5页Journal of Chongqing Medical University

摘　　要：目的:明确骨髓多能成体祖细胞(Bonemarrowmultipotentadultprogenitorcells,MAPCs)体外向心肌特异性分化及其与八聚体结合转录因子4(Octamer-bindingtranscription factor-4,Oct-4)表达的相关性。方法:将大鼠骨髓中分离出的MAPCs,在无血清培养基中连续培养35 d,采用5-氮杂胞苷(5-azacytidine,5-aza,1μmol/L)诱导24 h促使其向心肌细胞分化。结果:MAPCs在5-aza诱导的24 h后早期心脏特异性基因NKx2.5与GATA-4的mRNA水平显著增加,Western blot和免疫荧光染色显示心肌特异性蛋白Connexin-43与肌钙蛋白Ⅰ在5-aza诱导后的7 d开始表达,流式细胞分析表明在35 d后超过37%的细胞为肌钙蛋白Ⅰ阳性;同时实时定量聚合酶链反应、Western blot、免疫荧光染色和流式细胞技术检测到未分化的MAPCs高表达特异性干细胞标志物Oct-4,但在心肌分化过程中其表达明显下降。结论:MAPCs在5-aza诱导后能够有效地分化为心肌样细胞,并与Oct-4的表达下调相关。Objective：To investigate whether bone marrow multipotent adult progenitor cells（MAPCs）undergo cardiac specification and its dependablity with octamer-binding transcription factor-4（Oct-4）expression during their cardiomyocyte differentiation in vitro.Methods：MAPCs were isolated from rat bone marrow,treated with 5-azacytidine（5-aza,1 μmol/L）for 24 h,and cultured in a serum-free medium for cardiac differentiation for up to 35 days.Results：The cells started to express early cardiac-specific genes Nkx2.5 and GATA-4 with a significant increase in their mRNA level after 24h with 5-aza treatment.Western blot analysis and immunofluorescence staining revealed that the cardiac-specific proteins connexin-43 and troponin Ⅰ were expressed in the cells 7 days after 5-aza treatment.Flow cytometry analysis demonstrated that over 37% of the cells were positive for troponin Ⅰ by 35 days of differentiation,though the cells did not display spontaneous contraction.Meanwhile,the undifferentiated MAPCs expressed a significant level of the stem-cell-specific marker Oct-4 that was dramatically decreased in the cells shortly after the initiation of cardiomyocyte differentiation as evaluated using real-time（RT）-polymerase chain reaction,Western blot,immunofiuorescence staining,and flow cytometry.Conclusion：Downregulation of Oct-4 expression effectively accelerate cardiomyocyte differentiation of rat MAPCs after 5-aza induction in in vitro culture.

关 键 词：MAPCs OCT-4 大鼠 5-AZA 心肌细胞 细胞分化 

分 类 号：R322.11[医药卫生—人体解剖和组织胚胎学]