丹酚酸B诱导骨髓间充质干细胞向心肌样细胞分化

Differentiation of marrow mesenchymal stem cells into cardiomyocyte-like cells induced by salvianolic acid B

目的:体外分离、培养、纯化大鼠骨髓间充质干细胞(MSCs),并研究中药单体丹酚酸B诱导MSCs向心肌样细胞分化.方法:用贴壁培养法分离、培养、纯化MSCs,免疫荧光法检测细胞抗原CD44和CD34.分别用含5μmol/L的5-氮胞苷(5-aza)和30mg/L丹酚酸B的低糖DMEM培养液培养MSCs24h后,RT-PCR法检测培养1,7,14和21d心肌特异性转录因子NKX2.5,GATA-4和心肌特异性蛋白α-actin mR-NA的表达,用免疫荧光法检测诱导后α-actin的表达情况.结果:获得纯化的MSCs,免疫荧光检测细胞表面标记CD44呈阳性,CD34呈阴性.诱导后细胞形态逐渐发生变化.RT-PCR显示NKX2.5和GATA-4 mRNA在MSCs有基础性的表达,而分别以5-aza和丹酚酸B处理24h后,NKX2.5和GATA-4 mRNA表达增强,7d时表达达高峰,以后维持在高水平,与空白对照组相比,有统计学差异(P<0.01).未诱导的MSCsα-actin mRNA表达呈阴性,丹酚酸B诱导后的第14日,出现α-actin mRNA的表达.结论:用贴壁培养法可获得纯化的MSCs,丹酚酸B可诱导MSCs向心肌样细胞分化.

AIM： Bone marrow mesenchymal stem cells （MSCs） were isolated from SD mice and were then induced by salvianolic acid B to differentiate into cardiomyocyto-like cells. METHODS： MSCs were isolated, cultured, purified in vitro by adherent method and identified by immunohistochemical detection of cell markers CIM4 and CD34. MSCs were treated with 5-azacytidine or salvianohc acid B for 1 d. The expression of NKX2.5, GATA-4 and ct-actin mRNA in the MSCs harvested at day 1, 7, 14 and 21 were analyzed with reverse transcription-polymerase chain reaction. The immunohistochemical method was used to detect the expression of ct-actin protein after induction. RESULTS ： The cultured cells were CIM4 positive and CD34 negative by immunohistochemical detection and could be identified as MSCs. The morphological features of the cells changed after treatment with salvianolic acid B. RT-PCR assay displayed that NKX2.5 and GATA-4 mRNA expressed weakly in the MSCs. One day after iduction by 5-azacytidine or salvianolic acid B, the expression of NKX2.5 and GATA-4 mRNA increased （compared with blank control cells, P 〈 0.01 ）, reached the peak level at day 7 after induction, and kept at high level during the process of cultivation. The expression of ct-actin mRNA was detected at day 14 after induction by 5-azacytidine or salvianolic acid B. CONCLUSION： Pure MSCs can be obtained by adherent method. Salvianolic acid B can induce the differentiation of MSCs into cardiomyocyto-like cells.