Myoblast cell lines are regarded as progenitors of muscle cells containing filament and myofibrils, are characteristic of proliferation, self-renewal and multipotency. And they are recognized to contribute to reveal mechanisms of animal muscle development in vitro. In this study, both of the enzyme digestion and different speed adherence protocols were used to isolate and purify Ujumqin sheep (Ovis aries) myoblast cells derived fromlongissimu dorsi. The growth state of myoblasts was obtained with CCK-8 method, and myogenic differentiation was detected using H&E staining and Real-time PCR. The results showed that the highly retractile and slightly round cells adhered to the wall of the flask, but a few cells presented small projections after the cells were seeded into the culture flask for 24 h. Many elongated and spindle-shaped cells appeared 48 h after incubation. The growth curve showed that myoblast cells were in normal growth state. The standard curve was almost S-shaped, and the logarithmic phase occursed after 3 days. Myoblasts proliferated rapidly from the 3 rd to 8 th day, and the proliferation slowed down after 8 days, the stationary phase started on the 9th day. After the induction of myoblasts in 5 days, many mononucleate myoblasts entered into the plateau phase, and long bamboo-shaped primary myoblasts (myotubule), with centrally located nuclei, were formed. Gradually, the myotubule positioned themselves in a regular parallel arrangement and formed multinucleate myotubules. H&E staining of the differentiated cells showed blue-stained nuclei, pink-stained cytoplasm, and multinucleate long bamboo-shaped primary myotubule. These data suggested that the cells differentiated and fused to form myotubules. The expressions of MSTN (myostatin), FST (follistatin), BTG2 (B-cell translocation gene 2) and BTG3 (B-cell translocation gene 3) during differentiation were detected using Real-time PCR. The expressions of MSTN and FST gradually decreased with the progression of differentiation, but the expression of FST was higher than that of MSTN (P<0.05). The expression of BTG3 in the myoblasts was significantly higher than that in the differentiated cells at the early stage of differentiation (P<0.01); however, the expression of BTG2 in the myoblasts on the 3 rd and 7 th days was markedly lower than that in the undifferentiated cells (P<0.05). The expression levels of BTG2 and BTG3 were inversely proportional, and this indicated that these genes played different roles in muscle development. These findings confirmed that the separated myoblasts were myogenic and could form myotubules.The results suggested that Ujumqin sheep muscle-derived myoblast cells have the ablities to develop into myobubes in differentiation process. BTG2 and BTG3 expressed at different levels indicate that both gene maybe play different role in skeletal muscle growth.%成肌细胞(myoblast cell)是指胞浆中含有肌丝的肌组织前体细胞,具有自我更新和促进肌纤维再生的能力,是体外研究肌肉发育调控机理的理想载体.本研究采取成年乌珠穆沁羊(Ovis aries)的背最长肌组织,结合使用胶原酶消化和差速贴壁两种方法分离纯化羊成肌细胞,并应用CCK-8法检测细胞生长曲线,苏木素-伊红染色与定量PCR检测成肌分化效果.结果显示,分离的细胞接种到培养瓶中24h后,高折光性亮圆形细胞开始贴壁,少量细胞有小突起.培养48 h后,出现大量长梭形细胞,部分细胞伸出突起呈星型,细胞胞浆丰富.细胞生长曲线显示,成肌细胞符合正常生长规律,细胞传至第三代后用于CCK-8试剂盒检测,经分析后发现细胞符合正常生长规律,生长曲线近似S形,3d后开始进入对数生长期,第3～8天增殖迅速,第8天后细胞增殖速度减缓,第9天基本进入平台期.成肌诱导5d后,大量成肌细胞开始从单个核期进入合体细胞阶段,融合成核位于中央的多核性长竹状初生肌管.随后逐渐规律性地沿一个方向平行排列,形成多核的肌管.定量PCR结果显示,MSTN(myostatin)、FST (follistatin)、BTG2 (B-cell translocation gene 2)与BTG3 (B-cell translocation gene 3)在成肌细胞分化过程中有不同程度表达,存在阶段特异性.MSTN与FST随分化时间推移表达量逐渐降低,但FST基因表达量显著高于MSTN基因(P＜0.05)；分化期成肌细胞的BTG3表达极显著高于未分化期(P＜0.01),而BTG2的表达则显著低于细胞未分化状态(P＜0.05).研究结果提示,本研究分离的羊成肌细胞具有肌源性,可分化为肌管；BTG2与BTG3两基因呈相反趋势表达,可能说明基因在肌肉发育过程中起不同作用.
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