条件培养液体外诱导骨髓间充质干细胞向心肌样细胞分化的实验研究*

Experimental study on differentiation of bone marrow mesenchymal stem cells into myocardium-like cells induced by conditioned medium

关键词:  条件培养液  心复康  骨髓间充质干细胞  免疫细胞化学  实时荧光定量RT-PCR

Key words:  Conditioned medium  XinFuKang  MSCs  Immunocytochemistry  Real time fluorescent quantified RT-PCR

摘要:

[目的]观察乳鼠心室肌成纤维细胞条件培养液干预体外诱导骨髓间充质干细胞(MSCs)分化为心肌样细胞的作用及其机制。[方法]分离培养及鉴定大鼠MSCs,对其进行分组诱导:单纯条件培养液组(TJ)、条件培养液1组(TJ-ZY)、条件培养液2组(TJ-XFK),诱导,24h后继续培养4周,免疫细胞化学法鉴定心肌特异性肌钙蛋白T(cTnT)的表达,实时荧光定量逆转录-聚合酶链反应(RT-PCR)法检测心肌早期基因NKX2.5、GATA-4、Desmin和α-actinmRNA的表达。[结果]免疫细胞化学结果显示单纯条件培养液诱导后的MSCs不表达心肌特异性蛋白cTnT,心复康参与组则表达。实时荧光定量(RT-PCR)结果显示各组均表达心肌分化早期基因,心复康参与组诱导后的MSCs的心肌分化早期基因表达明显高于单纯条件培养液组。[结论]微环境在MSCs向心肌样细胞的分化过程中起了重要作用,心复康能够提高MSCs向心肌样细胞的分化和特化作用。

Abstract:

[Objective] To observe the effect of conditioned medium in inducing the bone marrow mesenchymal stem cells (MSCs) differentiation into myocardium-like cells.[Methods] MSCs were cultured,purified and identified.They were induced by pure conditioned medium (TJ),conditioned medium 1 (TJ-ZY) and conditioned medium 2 (TJ-XFK) respectively.After that they were cultured for four weeks and evaluated the expression of cardiac troponin T by immunocytochemistry.The expression of the early gene of cardiac muscle NKX2.5,GATA-4,Desmin and a-actin mRNA was evaluated by real time fluorescent quantify RT-PCR.[Results] After inducing,the pure conditioned medium group doesn't express cTnT,but the Xinfukang group does.All groups expressed the four early gene of cardiac muscle,but the expression of the genes in the Xinfukang intervention groups was much more than that in the pure conditioned medium group.[Conclusion] Microenvironment plays an important role in the process of MSCs induced into myocardium-like cells,the Xinfukang can improve the differentiation and specialization of MSCs into myocardium-like cells.