体外证据不支持5-氮胞苷诱导骨髓基质细胞向心肌细胞分化

NO IN VITRO EVIDENCE CAN VERIFY THE NOVEL EFFECTS OF 5-AZACYTIDINE ON CARDIOMYOGENIC DIFFERENTIATION OF MARROW STROMAL CELLS

目的 探讨胞嘧啶类化学物质 5 氮胞苷是否可诱导体外培养的骨髓基质细胞 (marrowstromalcells ,MSCs)向心肌细胞分化。方法 用不同浓度 (3,5 ,10 μmol/L)的 5 氮胞苷以各种方法 (一次处理与反复处理 )诱导原代及传代MSCs。采用免疫荧光及WesternBlot技术检测心肌特异性肌球蛋白重链 (myosinheavychainα/ β ,MHCα/ β)及肌钙蛋白I (tro poninI,TnI)的表达 ;并用心室肌特异性肌球蛋白轻链 (ventricularmyosinlightchain 2 ,MLC2v)启动子 (2 5 0bp)控制的增强型蓝绿色荧光蛋白 (enhancedcyanfluorescentprotein ,ECFP)报告基因表达技术检测被诱导MSCs是否发生MLC2v的转录启动。上述检测技术的特异性和可靠性用培养的心肌细胞进行验证。结果 在本试验最长观察时间内 (30d) ,各种浓度及各种方法 5 氮胞苷诱导的MSCs培养物中未见细胞自发搏动、肌管形成、心肌特异性MHC和TnI表达 ;亦无MLC2v转录启动阳性细胞出现。结论 从转录启动到翻译后水平的证据均不支持体外 5 氮胞苷处理可诱导MSCs表达心肌特异性蛋白。

Objective To explore if 5-azacytineas a cytosine analogue can trigger the cardiomyogenic differentiation of marrow stromal cells (MSCs). Method The primary and passaged MSCs were exposed to different concentrations (3, 5 and 10μmol/L)of 5-azacytineusing differentmethods (single or repeat treatment). The expression of cardiac specific proteins of myosin heavy chain α/β (MHCα/β) and troponin I(TnI) was detected with immunofluorescence and Western Blot technique; and the transcription promotion of the ventricular myosin light chain 2 (MLC2v) was detected with the enhanced cyan fluorescent protein (ECFP) reporter gene under the transcriptional control of MLC2v promoter (250 bp). The specificity and reliability of the detection methods were technically confirmed with cultured rat cardiomyocytes as the positive control. Result During the longest observation time (30 d), neither spontaneously beating cells nor formation of myotubes were found. The expression of cardiac specific proteins and the positive cells with cyan fluorescence were never observed in MSCs of all treated groups. Conclusion No evidence from transcription promotion to post-translation levels supports the novel effects of 5-azacytidineon the cardiomyogenic differentiation of MSCs in vitro.