利用激光扫描共聚焦显微镜实时监测体外诱导分化的人骨髓间充质干细胞内游离Ca^2＋浓度动态变化  

Real-time monitoring the dynamic change of intracellular free calcium concentration of differentiated human mesenchymal stem cells in vitro by laser scanning confocal microscope

作　　者：邓方阁[1] 郭连峰 张秀英[1] 李玉林[1] 

机构地区：[1]吉林大学白求恩医学院病理生物学教育部重点实验室,长春130021 [2]北京奥林巴斯销售服务有限公司生命科学事业部

出　　处：《中华生物医学工程杂志》2008年第5期354-357,共4页Chinese Journal of Biomedical Engineering

基　　金：国家高技术研究发展计划863计划（2004AA205020）

摘　　要：目的利用激光扫描共聚焦显微镜（LSCM）实时监测由人骨髓间充质干细胞（hMSC）诱导分化的心肌样细胞内游离Ca^2＋浓度的动态变化。方法体外分离培养hMSC，5-氮胞苷诱导向心肌样细胞分化。分别以体外分离培养的原代搏动的出生2dSD大鼠乳鼠心肌细胞（CM）和同代次的hMSC为阳性对照和阴性对照。Fluo-3／AM荧光探针分别标记hMSC、由hMSC诱导的心肌样细胞及CM，利用LSCM实时监测单细胞内Ca^2＋动态变化的优势，观察加入心脏正性肌力药物后的细胞反应及细胞内Ca^2＋水平的动态变化。结果静息状态下（给药前），搏动的CM荧光强度有变化，而hMSC及心肌样细胞Ca^2＋的荧光强度保持在稳定的波动水平内，两者差异无统计学意义。给药后，hMSC的Ca^2＋离子流没有改变，与静息状态下趋于一致；而心肌样细胞则表现出一个明显的Ca^2＋离子流，且下降后细胞内Ca^2＋水平明显高于加药前静息钙水平，其Ca^2＋离子流的表现趋势与搏动的CM基本一致。比较给药后hMSC与心肌样细胞的Ca^2＋荧光强度落差值，差异具有统计学意义（64．04±22．68比180．75±34．04，P〈0．01）。结论hMSC经5-氮胞苷诱导后，在电学特性方面具有向CM分化的趋势，心肌样细胞与CM具有某些相同的电生理特性。Objective To monitor in real-time the dynamic change of intracellular free calcium concentration of cardiomyo-like cells differentiated from human marrow mesenchymal stem cells （hMSCs） by laser scanning confocal microscopy （LSCM）. Methods Isolated and cultured hMSCs were induced into cardiomyo-like cells by 5-Aza in vitro. The same generation hMSCs with the induced hMSCs acted as negative control group, and the primary beating cardiomyocytes （CMs） isolated and cultured in vitro which derived from 2-day SD rats acted as positive control group, hMSCs, cardiomyo-like cells induced from hMSCs and CMs were dyed with Fluo-3/AM respectively. Utilizing the advantage of real time monitoring the dynamic change of calcium in unicell by LSCM, the cells response and intracellular dynamic change of calcium level in three kinds of cells were detected ＂after administration of positive inotropic heart drug. Results Under quiescent condition （before administration） , the fluorescence intensity changed in beating CMs, but remained in stable fluctuating level in either hMSCs or cardiomyo-like cells, and there was no significant difference in later two groups. After administration, the calcimn ion current of hMSCs did not change, which was as same as that in the quiescent condition. However, the calcium ion current of cardiomyo-like cells displayed an evident upgrade,and intracellular calcium level was higher than that in the quiescent condition after ion current descent, which was as same as that of CMs. Compared the fluctuating level of fluorescence intensity in hMSCs and cardiomyo-like ceils after administration, there was significant difference （64.04 ± 22.68 vs 180.75 ± 34. 04, P 〈 0.01 ）. Conclusions hMSCs induced by 5-Aza has the tendency of differentiation into CMs in the electrophysiology characteristics. Some electrophysiologic characteristics of cardiomyo-like cells are the same as those of CMs.

关 键 词：激光扫描共聚焦显微镜 间质干细胞 电生理学 心肌样细胞 游离CA^2+浓度 离子流 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]