小鼠肌源性干细胞高表达CD34、Sca-1、Bcl-2和desmin

High expression of CD34,Sca-1,Bcl-2 and desmin proteins in mouse muscle-derived stem cells

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。

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 doubling 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 observations of FCM and imnmnofluoreseence showed that the phenotypic characteristics of MDSCs were antibody - positive for CD34, Sea - 1, Bcl - 2 and desmin （ 〉 90% ）. With increasing the level of cell purifieation, 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 ean effectively isolate MDSCs. MDSCs express the antigens of CD34, Sea - 1, Bcl - 2 and desmin at high levels, and the 4 proteins can be used to identi- fy MDSCs. With a high proliferating ability in vitro, MDSCs are ideal seed cells for tissue engneering.