5-氮胞苷体外诱导骨髓间充质干细胞向心肌样细胞分化:超顺磁性氧化铁颗粒的标记

Differentiation of superparamagnetic iron oxide-labeled bone marrow mesenchymal stem cells into cardiomyocytes-like cells in vitro induced by 5-azacitidine

背景:骨髓间充质干细胞体外经多种诱导剂均可成功转化为心肌样细胞,目前国内外报道中普遍采用的诱导剂为5-氮胞苷。超顺磁性氧化铁颗粒直径小,可被干细胞吞噬,加上其顺磁性可被MRI探测到信号,因而成为目前广泛应用于活体检测干细胞移植的理想示踪剂。目的:与未经标记的干细胞相比,观察超顺磁性氧化铁颗粒标记方式对干细胞体外经5-氮胞苷诱导向心肌样细胞分化效应的影响。方法:分离培养猪骨髓间充质干细胞,实验组采用P3代细胞经超顺磁性氧化铁颗粒标记,标记后细胞经5-氮胞苷体外诱导24h后继续培养,对照组直接经5-氮胞苷诱导。2周后用抗结蛋白、肌球蛋白重链、心肌特异性肌钙蛋白I、连接蛋白43进行免疫细胞化学染色鉴定,经普鲁士蓝染色观察细胞内铁颗粒。结果与结论:5-氮胞苷诱导分化后细胞表达抗结蛋白、肌球蛋白重链、心肌特异性肌钙蛋白I和连接蛋白43,且超顺磁性氧化铁颗粒标记诱导细胞内普鲁士蓝染色呈阳性。提示超顺磁性氧化铁颗粒标记骨髓间充质干细胞经5-氮胞苷体外诱导后可以分化为心肌样细胞。

BACKGROUND：Bone marrow mesenchymal stem cells（BMSCs） induced by various inducers can differentiate into cardiomyocyte-like cells in vitro.The common inducer was 5-azacitidine.Superparamagnetic iron oxide（SPIO） particle has small diameter and can be phagocytized by stem cells.Its paramagnetism can be detected by magnetic resonance imaging（MRI）.Thus,SPIO has been an ideal tracer of transplanted stem cells in living bodies.OBJECTIVE：To observe the effects of differentiation of SPIO-labeled stem cells into cardiomyocytes-like cells in vitro induced by 5-azacitidine,and compare with unlabeled stem cells.METHODS：Pig BMSCs were isolated and cultured.In the experimental group,the third passage of BMSCs were marked with SPIO,and then 5-aza was added in vitro for 24 hours.In the control group,BMSCs were induced by 5-azacitidine directly.2 weeks later,the induced cells were evaluated with desmin,myosin heavy chain,cardiac troponin I and connexin-43 by immunocytochemical staining.Iron particles were detected by Prussian blue staining.RESULTS AND CONCLUSION：Desmin,myosin heavy chain,cardiac troponin I,connexin-43 were expressed in BMSCs induced by 5-azacitidine.Prussian blue staining showed numerous blue-stained iron particles in the cytoplasm of SPIO marked BMSCs.These indicate that SPIO-marked BMSCs can differentiate into cardiomyocytes-like cells under 5-azacitidine induction in vitro.