Transcriptomic Analysis of Differentiating Primary Myoblasts treated or not with TGFB1 recombinant protein or ITD-1 compound.. Transcriptomic Analysis of Differentiating Primary Myoblasts treated or not with TGFB1 recombinant protein or ITD-1 compound.

Transforming Growth Factor beta (TGFβ) signaling cascade plays a fundamental role during muscle development and regeneration. Previous reports proposed several roles for the TGFβ superfamily members and generally they are considered negative regulators of myogenic differentiation. However, the activation of this signaling pathway has been shown to be essential to obtain a correct muscle tissue repair process. Due to these contrasting results, we decided to investigate the role of TGFb cascade in muscle cells. To identify the genetic networks regulated by TGFB signaling in muscle cells, we performed transcriptome analysis of differentiated primary myocytes, stimulated with either TGFB1 or ITD-1 Overall design: We cultured primary myoblasts at 30,000 cells per cm2 onto matrigel-coated plates. Once adherent, cells were incubated in differentiation medium (DMEM with 2% Horse serum and 1% penicillin/streptomycin) for 24 hours. At this point, myoblasts were treated with TGFB1 recombinat protein at a final concentration of 20ng/ml or ITD-1 compound at 5mM for 24 hours.

转化生长因子β（Transforming Growth Factor beta，TGFβ）信号级联反应在肌肉发育与再生过程中发挥着基础性作用。既往研究提出了TGFβ超家族成员的多项功能，通常认为其为肌源性分化（myogenic differentiation）的负调控因子。然而，已有研究显示该信号通路的激活对于完成正常的肌肉组织修复过程不可或缺。鉴于这些相互矛盾的实验结果，我们决定探究TGFβ级联反应在肌肉细胞中的作用。为鉴定肌肉细胞中受TGFβ信号通路调控的遗传网络，我们对经TGFβ1或ITD-1刺激后的分化原代肌细胞开展了转录组分析（transcriptome analysis）。
总体实验设计：我们将原代成肌细胞以每平方厘米30000个细胞的密度接种于基质胶（matrigel）包被的培养板中。细胞贴壁后，置于分化培养基（含2%马血清与1%青霉素/链霉素的DMEM培养基）中培养24小时。此时，将成肌细胞分别用终浓度为20ng/ml的重组TGFβ1蛋白，或5mM的ITD-1化合物处理24小时。