成肌细胞外泌体诱导骨髓间充质干细胞成肌分化的实验研究

Study of myoblast-derived exosomes in inducing myogenic differentiation of bone marrow mesenchymal stem cells

目的探索成肌细胞外泌体诱导骨髓间充质干细胞成肌分化的可行性。方法大鼠骨骼肌卫星细胞经体外培养成肌分化,提取成肌细胞外泌体与骨髓间充质干细胞共孵育,诱导其向骨骼肌分化,检测成肌分化以及各生长因子分泌变化。结果通过酶消化分离、差速贴壁提取的肌卫星细胞,Pax7表达阳性,增殖、分化后Myogenin、Desmin及Myosin表达阳性,Desmin流式细胞鉴定阳性率为94.5%。超速离心法提取到圆形或椭圆形的成肌细胞外泌体,外形呈杯状,磷钨酸负染;WB检测CD9、CD63、CD81及TSG101表达阳性。GFP标记的外泌体可被骨髓间充质干细胞摄取。外泌体与骨髓间充质干细胞共同孵育,3 d后Myogenin表达阳性,6 d后Desmin与Myosin表达阳性。ELASA检测细胞上清胰岛素样生长因子Ⅰ(insulin like growth factor,IGFⅠ)、肝细胞生长因子(heptocyte growth factor,HGF)及成纤维生长因子(fibroblast growth factor2,FGF2)水平明显升高。结论成肌细胞外泌体在体外能诱导骨髓间充质干细胞向成肌分化,刺激各种生长因子分泌可能是外泌体促进细胞增殖、分化的机制之一。

Objective To explore the feasibility of myoblast-derived exosomes(MB-Exos)in inducing myogenic differentiation of bone marrow mesenchymal stem cells(BM-MSC)in vitro.Methods Rat skeletal muscle satellite cells were differentiated into myoblasts.And MB-Exos were extracted and co-incubated with BM-MSC for inducing myogenic differentiation.The expressions of myogenin,desmin and myosin were measured and the level of growth factors after exosome inducing was detected.Results There was a positive expression of Pax7 and myogenic differentiation was detected.The purification rate of myoblast cells was 94.5%under fluorescence-activated cell sorting(FACS).Vesicle-like morphology of exosomes was visualized via transmission electron microscope(TEM)after an extraction of gradient ultracentrifugation.With a typical cup shape,the diameter was 30 to 200 nm with a negative stain of phosphotungstic acid.Western blot revealed that exosome markers of CD9,CD63,CD81 and TSG101 were expressed in MB-Exos.GFP-labeled MB-Exos were ingested by BM-MSC.Under the stimulation of MB-Exos,myogenic differentiation of BM-MSC was verified as an expression of myogenin in 3 days and an expression of desmin and myosin in 6 days.The levels of IGF-1,HGF and FGF2 in supernatant of cultured BM-MSC spiked markedly according to enzyme-linked immunosorbent assay(ELISA).Conclusions MB-Exos induces myogenic differentiation of BM-MSC in vitro and promotes the secretion of various growth factors.It may be one of the mechanisms of exosomes promoting cellular proliferation and differentiation.