淫羊藿苷诱导小鼠胚胎干细胞体外定向分化为心肌细胞方法学研究

Study on Induction of Icariin on Directional Differentiation of Embryonic Stem Cells into Cardiomyocytes in Vitro

目的采用药物淫羊藿苷(icariin,ICA)改变小鼠胚胎干细胞(embryonic stem cell,ES细胞)体外培养的微环境,建立一种药物介导提高ES细胞体外定向心肌细胞分化率的实验体系。方法采用悬滴培养法,构建ICA诱导ES细胞体外定向分化为心肌细胞的实验评价体系;并利用RT-PCR法和免疫荧光法鉴定心肌细胞。结果ICA在10-7mol.L-1浓度时,对ES细胞定向分化为心肌细胞呈现最佳诱导效应,分化率最高达84%(P<0.001)。ES细胞源心肌细胞表达心肌特异性基因肌球蛋白重链(α-MHC)和心室肌球蛋白轻链(MLC-2v),且心肌特异性肌小节α辅肌动蛋白(α-actinin)和肌钙蛋白T(troponin T)呈阳性表达。结论悬滴培养法结合药物改善微环境,可用于药物诱导ES细胞定向分化心肌细胞的实验体系。

OBJECTIVE To evaluate the method of inducing the directional differentiation of embryonic stem （ES） cells into cardiomyoeytes using icariin （ICA） in vitro. METHODS With Hanging-drop cultures of ES cells, ICA was used as an inducer of the directional differentiation of ES cells into cardiomyocytes. The expressions of cardiac-specific genes and sarcomeric proteins were verified by semi-quantitative RT-PCR and immunocytoehemistry analysis. RESULTS ICA at 10-7mol · L^-1 significantly enhanced cardiac differentiation,and up to 84% of the beating embryoid bodies （EBs） was found. The expressions of α-cardaic MHC and MLC-2v genes were detected in cardiomyocytes derived from EBs on early differentiation stage. Furthermore, the differentiated beating cardiac cells were stained positively with anti-α-actinin mAb and anti-troponin T mAb. CONCLUSION An effective method was established by combining drug with hanging-drop cultures of ES cells for inducing the differentiation of ES cells into cardiomyocytes in vitro.