体外心肌梗死微环境下大鼠骨髓间充质干细胞向内皮样细胞的分化

背景：间充质干细胞来源于中胚层,具有多向分化的能力。有研究显示骨髓间充质干细胞有向内皮细胞分化的能力,从而有助于心肌梗死后心脏新生血管的形成和心肌修复。 目的：观察骨髓间充质干细胞体外成内皮样细胞分化的特点,以及体外模拟心肌梗死微环境对骨髓间充质干细胞向内皮样细胞分化的影响。 方法：骨髓取自SD大鼠股骨和胫骨,骨髓分离培养骨髓间充质干细胞,体外扩增,对其形态学、增殖能力及多向分化能力进行鉴定;建立大鼠心肌梗死模型,制备8 h、3 d、1周、2周、4周梗死后不同时间点心肌组织的匀浆,分别加入到由血管内皮生长因子、碱性成纤维细胞生长因子β、胰岛素样生长因子1组成的内皮细胞诱导体系中,共同诱导2周后,观察不同时间的心肌组织匀浆对骨髓间充质干细胞增殖和成内皮分化的影响,检测vWF特异性抗原及表达的阳性率。 结果与结论：通过对培养细胞的形态学及功能鉴定,证明其为骨髓间充质干细胞,并具有成骨、成脂肪细胞、成内皮样细胞分化的能力。加入生长因子诱导体系诱导后的细胞部分表达vWF,梗死心肌匀浆与生长因子共同作用,能促进骨髓间充质干细胞向内皮细胞分化,vWF阳性率提高(P < 0.05)。RT-PCR显示加入梗死1周心肌组织匀浆诱导的骨髓间充质干细胞其vWF阳性表达率最高。提示骨髓间充质干细胞是一种具有多向分化能力的有别于造血干细胞的另一种骨髓干细胞。骨髓间充质干细胞具有向内皮分化的能力,且加入梗死心肌组织匀浆的诱导体系在体外可促进骨髓间充质干细胞向内皮细胞的分化。应用骨髓间充质干细胞治疗的最佳时机在1周为宜。

Differentiation of rat bone marrow mesenchymal stem cells into endothelioid cells in myocardial infarction micro-environment in vitro

BACKGROUND:Derived from the mesoderm, bone marrow mesenchymal stem cells (BMSCs) have the ability of differentiation into a variety of tissues. BMSCs can differentiate into endothelial cells under certain conditions to improve regeneration of blood vessel and repair of injured myocardium. OBJECTIVE:On the bases of establishment of BMSCs cultural system, myocardial infarction (MI) model and differentiating system into endothelial, this study was going to investigate the characters of BMSCs differentiating into endothelial cells and to initially observe the influence of infarcted myocardial micro-circumstances on BMSCs differentiation into endotheliod cells in vitro. METHODS:The bone marrow was extracted from the thighbone and tibiae of SD rats. BMSCs was isolated from rat marrow, cultured and expanded in vitro. Through evaluating the change of morphology, ability of multiplication and differentiating abilities into osteoblast and adipocytes, BMSCs were identified. After a rat MI model was made and myocardial tissue extract of different time after MI: 8 hours, 3 days, 1 week, 4 weeks was prepared, these different tissue extract was combined with the endothelial inducing system including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and insulin growth factor-1 (IGF-1) to induce BMSCs. After 2 weeks, the influence of different inducing circumstances on BMSCs expansion and differentiation into endothelium was observed. Special antigen of Von Willarbrand factor (vWF) was tested at the same. Main outcome measures include: (1)The shapes of the cells, growth curves and the abilities of differentiating into osteoblast and adipocytes were observed; (2)Induced cells:①growth curves; ②the expression rate of the antigen vWF through immunohistochemical; ③RT-PCR method used to test vWF-mRNA expression through semi-quantitative analysis. RESULTS AND CONCLUSION:It was confirmed that the cultured cells was BMSCs and could differentiate into osteoblasts, adipocytes and endotheliod cells. Partial BMSCs induced by growth factors showed positive of vWF and infarcted myocardium tissue extract have cooperative effect with these factors that could improve the process of BMSCs differentiating into endothelial cells (P < 0.05). RT-PCR showed BMSCs induced by infarcted myocardial tissue extract of 1 week could improve this differentiation into endothelium had the highest vWF positive rate. BMSCs are another kind of marrow stem cells apart from hematopoietic stem cells and have ability of multi-potential differentiation into many kind of lineage including endothelium. The induce system contained infarcted myocardium tissue extract could improve BMSCs differentiating into endothelial cells in vitro. This ability may cause BMSCs to play an important role in the repair of injured myocardium and may be the major mechanism of improving the cardiac function during cell therapy. The optimal time of cell therapy is probably at 1 week after MI.