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目的通过改良差速贴壁法分离纯化绵羊MDSC,探索转化生长因子(TGF-β1)诱导绵羊肌源性干细胞分化为平滑肌的可能性。方法取成年绵羊股四头肌细胞采用改良差速贴壁法(modified preplate technique)进行优化、分离、纯化获得绵羊级源性干细胞(MDSC)。利用流式细胞仪鉴定CD44、CD34、CD31、CD45、CD14的表达;western bloting法检测干细胞标志物desmin表达情况鉴定MDSC。然后利用10 ng/ml TGF-β1诱导MDSC向平滑肌方向分化;随后利用Western bloting法检测诱导培养基处理的MDSC中平滑肌蛋白标志物a-SMA和calponin的表达;结果通过对改良差速贴壁法成功分离出成年绵羊的肌源性干细胞(MDSC)。流式细胞仪鉴定其干细胞标志物CD44、CD34阳性表达,同时CD31、CD45、CD14表达阴性。Western blotting检测MDSC中Desmin呈阳性表达。利用10 ng/ml TGF-β1成功诱导MDSC向平滑肌方向分化。诱导过程中,平滑肌特异蛋白a-SMA和calponin在诱导细胞中的表达逐步上升。结论本研究通过对差速贴壁法成功分离出成年雌性绵羊的肌源性干细胞(MDSC)。并首次利用TGF-β1诱导羊肌源性干细胞(MDSC)成功向平滑肌方向分化。
OBJECTIVE: To isolate and purify sheep MDSCs by modified differential adherence method and to explore the possibility of transforming growth factor-beta (TGF-β1) -induced muscle myogenic stem cells differentiating into smooth muscle. Methods Sheep quadriceps femoris cells were modified by modified preplate technique, and sheep-derived stem cells (MDSCs) were isolated and purified. The expression of CD44, CD34, CD31, CD45 and CD14 was identified by flow cytometry. The expression of stem cell marker desmin was detected by western blotting to identify MDSC. Then, MDSCs were induced to differentiate into smooth muscle by using 10 ng / ml TGF-β1. Subsequently, the expression of a-SMA and calponin in MDSCs treated with induction medium was detected by Western blotting. Adult sheep myogenic stem cells (MDSCs) were successfully isolated. Flow cytometry identified its stem cell markers CD44, CD34 positive expression, while CD31, CD45, CD14 negative expression. Western blotting detected Desmin positive expression in MDSC. MDSCs were successfully induced to differentiate into smooth muscle by 10 ng / ml TGF-β1. During induction, the expression of smooth muscle specific proteins a-SMA and calponin in induced cells gradually increased. Conclusion This study successfully isolated adult female sheep myogenic stem cells (MDSCs) by differential adherence method. For the first time, TGF-β1 was used to induce myogenic myeloid stem cells (MDSCs) to differentiate into smooth muscle.