5-氮胞苷与HGF因子体外联合诱导人骨髓间充质干细胞诱导分化为心肌样细胞实验探讨

Study on Induce and Differentiation of Mesenchymal Stem Cells into Cardiomyocyte-like cells by 5-Aza and HGF

目的探讨5-氮胞苷(5-Aza)与肝细胞生长因子(HGF)作为诱导剂,对人骨髓间充质干细胞(hMSCs)分化为心肌样细胞的影响。方法采用密度梯度培养法与贴壁培养法相结合分离培养hMSCs,取第3代hMSCs随机分为3组,分别为HGF因子诱导分化组、5-Aza诱导分化组、HGF因子与5-Aza联合诱导分化组,诱导后培养4周,采用免疫细胞化学法对分化的心肌样细胞进行鉴定。结果结果表明hMSCs贴壁呈集落生长,有成纤维细胞样外观。各组诱导后细胞均呈梭形,排列方向渐趋一致,有肌管样结构形成。免疫细胞化学法检测心肌特异性蛋白结蛋白(Desmin)、心肌肌钙蛋白I(cTnI)表达阳性,未经诱导的同培养天数的hMSCs中均未见表达。HGF不能诱导hMSCs向心肌细胞转化,但HGF因子与5-Aza联合诱导分化组心肌样细胞分化率明显高于其他两组。结论 hMSCs体外在HGF诱导下可定向分化为心肌样细胞,5-Aza与HGF体外联合应用可以提高hMSCs的诱导分化效率。

Objective To explore the characteristics of differentiation of hMSCs into cardiomyocyte-like ceils and expression of Desmin, cTnI proteins in cardiomyocyte-like cells by using 5-Aza and HGF in vitro. Meth- oils The bone marrow was derived from human sternum, and hMSCs were isolated and cultured by the methods of density gradient centrifugation and adherent filtration. The hMSCs to three groups, then they were treated with5-Aza, HGF and 5-Aza of third passage were randomly divided in- ＋ HGF. The differentiation of hMSCs was examined with immunocytochemical staining after 4 weeks. Results The hMSCs showed adherent growth, formed colonies, and had fibroblast-like morphology in vitro. The cells had spindle-like shape, arranged in same direction, formed myotube-like structures, and expressed Desmin and cTnI proteins in all groups. HGF could not induce and differentiate hMSC into cardiomyocyte-like cells. The Desmin and cTn I positive rate in the HGF with 5- Aza group were significantly higher than other two groups （ P 〈 O. 05 ）. Conclusion The hMSCs can be induced and differentiated into cardiomyocyte-like cells by using HGF and 5-Aza.