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目的:探讨丹参单体丹酚酸B(Salvianolic acid B,SalB)在体外诱导大鼠骨髓间充质干细胞(bone marrowmes-enchymal stem cells,MSCs)分化为心肌样细胞的情况及Nkx2.5、GATA-4 mRNA的表达,从而确定大鼠MSCs体外诱导为心肌样细胞的条件、规律及转化细胞的特点。方法:选用成年近交系Wistar大鼠,利用percoll分离液进行密度梯度离心法和直接贴壁法进行分离、提纯MSCs,并进行培养扩增。免疫组化方法对第9代MSCs表面抗原进行鉴定。分别应用10μmol/L5-氮胞苷(5-azacytidine,5-aza)及250μg/L SalB联合5-aza(5-aza+SalB)对第9代的MSCs进行联合诱导24h,于诱导后第4周,用实时荧光定量RT-PCR法检测GATA-4和Nkx2.5基因表达。结果:①第9代MSCs免疫组化染色CD44呈阳性表达,CD34呈阴性表达。②诱导4周后,细胞体积变小,可见由几个细胞连接形成的多核肌管样结构。5-aza组、5-aza+SalB组均出现明确的心肌早期分化基因与5-aza组相比,5-aza+SalB组的NKx2.5、GA-TA-4 mRNA表达明显增加。结论:SalB具有促进5-aza诱导的MSCs向心肌样细胞分化的作用,并且在Nkx2.5基因表达上丹酚酸B的优势更加明显。
Objective: To investigate the differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) into cardiomyocyte-like cells induced by Salvianolic acid B (SalB) and Nkx2.5, GATA The expression of -4 mRNA was used to determine the conditions, regularity, and characteristics of transformed cells in which rat MSCs were induced into cardiomyocyte-like cells in vitro. METHODS: Adult inbred Wistar rats were selected and percoll separation solution was used for density gradient centrifugation and direct adherence method to separate and purify MSCs. Immunohistochemical methods were used to identify the surface antigens of the 9th generation MSCs. 10μmol/L 5-azacytidine (5-aza) and 250μg/L SalB combined with 5-aza (5-aza+SalB) were used to induce the 9th generation of MSCs for 24h, respectively, after induction. Zhou, GATA-4 and Nkx2.5 gene expression were detected by real-time fluorescence quantitative RT-PCR. RESULTS: The expression of CD44 in the 9th passage MSCs was positive and CD34 was negative. 2 After 4 weeks of induction, the cell volume became smaller, and a multinuclear myotube-like structure formed by the connection of several cells was observed. In the 5-aza group and the 5-aza+SalB group, there was a clear early differentiation of the myocardium. Compared with the 5-aza group, the expression of NKx2.5 and GA-TA-4 mRNA in the 5-aza+SalB group was significantly increased. Conclusion: SalB can promote the 5-aza-induced differentiation of MSCs into cardiomyocyte-like cells, and the advantage of salviandic acid B in Nkx2.5 gene expression is more obvious.