|
|
|
Abstract 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 from longissimu 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 refractile 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.
|
|
Received: 26 October 2011
|
|
|
|
|
|
