兔肌源性干细胞的生物学特性及表型研究

Isolation and characterization of rabbit muscle-derived stem cells

目的:探讨兔肌源性干细胞(MDSCs)的分离及特征和表型研究。方法:取1.5月龄新西兰兔大腿肌肉,采用差速贴壁分离Prep late技术,分别用Ⅺ胶原酶、d ispase蛋白酶以及胰蛋白酶分步消化肌肉,并用差速贴壁法分离晚期贴壁的细胞,流式细胞仪(FCM)、免疫细胞化学以及W estern b lotting等检测初步确定细胞表型。结果:进行连续6 d的差速贴壁分离。第3 d开始得到较多量的圆形或短梭形细胞,簇样生长,体积明显小于骨骼肌细胞及其它杂细胞,10 d左右汇合,有极强的融合生长倾向。细胞在汇合度<30%传代可较好地保持原形态,流式细胞仪检测示MDSCs>80%为desm in+,>70%为Bc l-2+,>95%为CD45-,免疫细胞化学定性示desm in+。W estern b lot-ting检测显示随着细胞的纯化,α-SMA表达越来越弱。而细胞高汇合度(>50%)或低血清培养时极易融合成肌管或肌细胞链,skeletalmyosin+。结论:MDSCs具有desm in、Bc l-2高水平表达和CD45极低水平表达的特性,具有多向分化能力的MDSCs是组织工程研究的一种新型种子细胞。

AIM： To explore the feasibility of isolation and characterization of the muscle - derived stem cells （MDSCs） of rabbits. METHODS： Skeletal muscle specimens were harvested from rabbits of 1.5 months. Preplate technique was applied to isolate MDSCs, and flow cytometry （ FCM ）, immunocytochemistry and Western blotting were performed to characterize MDSCs. RESULTS： The isolating process contained 6 consecutive days of differentiated attachment. From day 3, many round - shaped or short spindly - shaped ceils began to appear, which were markedly smaller than the skeletal muscle cells and grew to confluence after about 10 d with obvious tendency to fuse. When passaged below confluence of 30%, these t：ells maintained their original shape well, and were shown 〉 80% desmin^＋ , 〉 70% Bcl -2^＋ , CD45^- by FCM. Desmin^＋ by immunocytochemistry and decreasing expression of α - SMA by WB were also observed. When passaged at high confluence （ 〉 50% ） or cultured with low concentration of serum, these cells tended to fuse to form myotubes, and were skeletal myosin^＋. CONCLUSION： Preplate technique can effectively isolate MDSCs, this provides tissue engineering with a new type of seed cells.