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首页> 外文学位 >Stretch-induced effects on microRNA expression and exogenous microRNA delivery in differentiating skeletal myoblasts.
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Stretch-induced effects on microRNA expression and exogenous microRNA delivery in differentiating skeletal myoblasts.

机译：拉伸诱导的分化骨骼肌成肌细胞中的microRNA表达和外源microRNA传递的影响。

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摘要

The research presented here represents a quest to understand and address limitations in the field of skeletal muscle tissue engineering, with hopes to better understand the factors involved in producing viable, engineered tissues. The driving force behind this research was to address two of the many factors important in muscle cell proliferation and differentiation, toward developing mature and functional bioartificial skeletal muscles (BAMs). Our work focused on understanding the individual effects of mechanical stimulation and microRNAs (miRNAs), as well as the synergistic relationship between the two factors. We hypothesized that (1) myoblast proliferation and differentiation are modulated by mechanical stimulation via temporally regulated miRNAs and that (2) modulating these miRNAs can enhance skeletal muscle function in a 3D tissue-engineered system.;We first established a BAM system using C2C12 mouse myoblasts in a collagen gel, showing that these cells were able to produce mature sarcomeres when cultured under steady, passive tension for up to 36 days. Staining muscle-specific proteins and electron microscopy showed distinct striations and myofiber organization as early as 6 days, post-differentiation. At 33 days, cultures contained collagen fibers and showed localization of paxillin at the fiber termini, suggesting that myotendinous junctions were forming.;We then focused on the effects of mechanical stimulation on C2C12 myoblasts in a simpler 2D system. In particular, miRNA and muscle-specific gene expression were assessed over time and in response to two cyclic stretch regimens using miRNA microarray technology and quantitative real time RT-PCR. Both miRNAs and certain genes, such as Mef2c, had differential responses to the two regimens. Over-expression and inhibition studies of one muscle-specific miRNA, miR-1, abrogated the stretch response, suggesting that a balancing mechanism is in place to avoid large fluctuations in miRNA levels.;Finally, since miRNA modulation quenched the stretch-mediated response in myoblasts, we chose to examine 3D BAM function when miRNA levels were altered to promote differentiation. Using the same collagen gel model established previously, a muscle-specific miRNA, miR-133, known to promote proliferation, was transiently inhibited (anti-miR-133) to encourage differentiation. Forces in the anti-miR-133 BAMs were, on average, 20% higher over the negative control. Further, myofiber diameters were significantly greater, and striations were more organized in the anti-miR-133 BAMs, suggesting that transient, exogenous delivery of miRNAs may be a viable approach to create a more fully differentiated muscle.
机译：此处提出的研究代表了对骨骼肌组织工程领域的理解和解决的一项探索，希望能更好地了解生产可行的工程组织所涉及的因素。这项研究背后的驱动力是解决在肌肉细胞增殖和分化中起重要作用的许多因素中的两个，以发展成熟和功能性的生物人工骨骼肌（BAM）。我们的工作重点是了解机械刺激和microRNA（miRNA）的个体作用，以及这两个因素之间的协同关系。我们假设（1）通过时间调控的miRNA通过机械刺激调节成肌细胞的增殖和分化，并且（2）调节这些miRNA可以增强3D组织工程系统中的骨骼肌功能。我们首先使用C2C12小鼠建立了BAM系统胶原蛋白凝胶中的成肌细胞，显示这些细胞在稳定，被动的张力下培养长达36天时能够产生成熟的肉瘤。染色后的6天之初，染色的肌肉特异性蛋白质和电子显微镜显示出明显的条纹和肌纤维组织。在第33天时，培养物中含有胶原纤维，并显示了帕西林在纤维末端的定位，这表明正在形成肌腱接头。然后，我们在简单的2D系统中集中研究了机械刺激对C2C12成肌细胞的影响。特别是，使用miRNA芯片技术和实时定量RT-PCR评估了miRNA和肌肉特异性基因的表达随时间推移以及对两种循环拉伸方案的反应。 miRNA和某些基因（例如Mef2c）对这两种方案都有不同的反应。一项针对肌肉的特定miRNA miR-1的过度表达和抑制研究消除了拉伸反应，这表明已经建立了一种平衡机制来避免miRNA水平的大幅波动。最后，由于miRNA的调节终止了拉伸介导的反应在成肌细胞中，当改变miRNA水平以促进分化时，我们选择检查3D BAM功能。使用先前建立的相同胶原蛋白凝胶模型，已知会促进增殖的肌肉特异性miRNA miR-133被短暂抑制（抗miR-133）以促进分化。抗miR-133 BAM的作用力平均比阴性对照高20％。此外，抗miR-133 BAMs的肌纤维直径明显更大，条纹更加有条理，这表明miRNA的瞬时，外源性传递可能是创建更完全分化的肌肉的可行方法。

著录项

  • 作者

    Rhim, Caroline.;

  • 作者单位

    Duke University.;

  • 授予单位 Duke University.;
  • 学科 Biology Molecular.;Engineering Biomedical.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 213 p.
  • 总页数 213
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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