AIM : To explore the characteristics and phenotype of the muscle - derived stem cells( MDSCs ) of mouse.METHODS: Skeletal muscle specimens were harvested from C57BL/6 mice of 2 weeks old.Preplate technique was applied to isolate MDSCs.The cellular growth status and morphology of the primary MDSCs were observed.The sphere - forming, proliferation and differentiation assays were performed and flow cytometry( FCM ). immunocytochemistry and Western blotting were used to characterize MDSCs.RESULTS : The isolating process contained 6 consecutive days of differentiated attachment.MDSCs were round or short spindle - shaped cells.The growth curve of MDSCs showed logarithmic growth, and the douhling time of MDSCs was( 9.69±2.08 ) h.Cloning efficiency of MDSCs was( 13.35 ± 2.54 )％ .When the cells at high confluence( ＞ 50％ ) or cultured with low concentration of serum( 2％ FBS ), they tended to fuse to form myotubes.The ohservations of FCM and immunofluorescence showed that the phenotypic characteristics of MDSCs were antibody - positive for CD34, Sca - 1, Bcl - 2 and desmin( ＞ 90％ ).With increasing the level of cell purification,the upregulation of desmin expression and the downregulation of α - SMA expression from preplate 1 cells( PP1 ) to preplate 6 cells( PP6 ) were observed by Western blotting.CONCLUSION : The preplate technique can effectively isolate MDSCs.MDSCs express the antigens of CD34, Sca - 1 , Bcl - 2 and desmin at high levels, and the 4 proteins can be used to identify MDSCs.With a high proliferating ability in vitro, MDSCs are ideal seed cells for tissue engineering.%目的:探讨小鼠肌源性干细胞(MDSCs)的生物学特性和表型.方法:取小鼠骨骼肌,用酶分步消化,应用差速贴壁法进行纯化获取MDSCs,观察细胞的形态,对MDSCs分别进行增殖能力、克隆形成率和融合情况等鉴定;通过流式细胞术、免疫荧光法及免疫印迹法(Western blotting)等检测初步确定细胞表型.结果:经差速贴壁法获取的MDSCs为圆形或短梭形细胞,聚集成簇,呈对数生长;细胞倍增时间(9.69±2.08)h,克隆形成率(13.35±2.54)%;细胞在高汇合度(＞50%)或低血清(2%FBS)培养时,极易融合成肌管或肌细胞链;流式细胞术和免疫荧光技术鉴定:CD34、Sca-1、Bcl-2和desmin在原代MDSCs中的表达均＞90%;Western blotting检测显示随着细胞的纯化, desmin表达越来越强,α-SMA表达越来越弱.结论:MDSCs通过差速贴壁法获取时纯度较高,它具有高水平表达CD34、Sca-1、Bcl-2和desmin的生物学特性,这4种蛋白可作为鉴定MDSCs的标志物.MDSCs增殖旺盛,可在体外大量扩增,是组织工程研究的一种新型种子细胞.
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