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Influence of mechanical cues and the extracellular matrix on patterns of cell migration and rates of proliferation

机译：机械提示和细胞外基质对细胞迁移模式和增殖速率的影响

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摘要

The mechanical environment of a cell and its tissue can impact multiple biological processes including development, wound healing, and metastasis. Specific cellular behaviors influenced by the mechanical microenvironment include differentiation, morphology, apoptosis, migration, and proliferation. In this thesis I have focused specifically on the effect of environmental stiffness and applied mechanical forces on cellular migration and proliferation, respectively. Using two different applications, both tailored to evaluate the mechanical forces alone on cellular behavior, I attempted to simulate the mechanical composition of the in vivo tissue microenvironments in vitro using polyacrylamide hydrogels. To test whether cells maintain a mechanical memory for a specific stiffness in vitro, we utilized a substrate that differentially polymerizes with variant levels of UV exposure and analyzed the directional migration patterns upon different rigidities. These substrates did not show any particular directional preference for migration, however cells did seem to be able to sense variation in stiffness based on the results of a morphology assay. It is unknown whether the cells were unable to sense differences in neighboring stiffnesses due to the extracellular matrix or to the hydrogel itself. To examine the proliferation rates of cells given an applied mechanical stimulus, we created hydrogels embedded with magnetic microbeads that provided a tugging and pulling motion mimicking the effects of adherent cells on their neighboring environment. The observed increase in proliferation upon mechanical stimulation was dependent on the presence of fibronectin coated to the hydrogel surface, indicating that this protein is essential for the mechanosensing response of cells. I hypothesized that compacted conformations of fibronectin are released during mechanical stimulation, opening cryptic binding sites for cells to adhere to. I tested the presence of these cryptic binding sites by chemically crosslinking the ECM prior to stimulation, as well as adding the competitive peptide arginine-glycine-aspartic acid (RGD). Both of these resulted in the decrease in proliferation rate during stimulation but had no effect in control cells. The surface receptor protein responsible for activating these cascades is still unknown. After testing the activity level of beta1 integrin, a known mechanosensor and binding partner to fibronectin, there was no difference in the activity of this particular integrin subunit, strongly suggesting this is not the integrin activated by our mechanical stimulus. Protein activity studies found that the phosphorylation state of both Focal Adhesion Kinase (FAK), as well as, Extracellular Signal-Regulated Kinase (ERK) are increased upon stimulation, indicating that these two signaling cascades lead to the increase in the cell cycle activity. Further studies are required to determine the link between the fibronectin cryptic sites and the downstream signaling cascades activated during stimulation. Both of these cell behavioral studies will help to better understand the extent of impact the mechanical environment has on living tissue systems.
机译：细胞及其组织的机械环境会影响多种生物学过程，包括发育，伤口愈合和转移。受机械微环境影响的特定细胞行为包括分化，形态，凋亡，迁移和增殖。在这篇论文中，我专门研究了环境刚度和施加的机械力分别对细胞迁移和增殖的影响。我使用两种不同的应用程序（都专门设计来单独评估对细胞行为的机械力），尝试使用聚丙烯酰胺水凝胶模拟体外活体内组织微环境的机械组成。为了测试细胞是否在体外针对特定的刚度保持机械记忆，我们利用了可以在不同水平的紫外线照射下差异聚合的基质，并分析了不同刚度下的定向迁移模式。这些底物对迁移没有显示任何特定的方向性偏好，但是根据形态分析的结果，细胞似乎确实能够感知到刚度的变化。尚不清楚细胞是否由于细胞外基质或水凝胶本身而无法感知相邻刚度的差异。为了检查在施加了机械刺激的情况下细胞的增殖速率，我们创建了嵌入磁性微珠的水凝胶，该微凝胶提供了拉扯和牵拉运动，模仿了粘附细胞对其周围环境的影响。在机械刺激下观察到的增殖增加取决于纤连蛋白包被在水凝胶表面的存在，表明该蛋白对于细胞的机械传感反应至关重要。我假设在机械刺激过程中释放了紧密结合的纤连蛋白构象，从而打开了细胞粘附的隐蔽结合位点。我在刺激之前通过化学交联ECM以及添加竞争性肽精氨酸-甘氨酸-天冬氨酸（RGD）来测试这些隐性结合位点的存在。这些都导致刺激期间增殖速率的降低，但对对照细胞没有影响。负责激活这些级联反应的表面受体蛋白仍是未知的。在测试了β1整联蛋白（一种已知的机械传感器和纤连蛋白的结合伴侣）的活性水平后，该特定整联蛋白亚基的活性没有差异，强烈表明这不是我们的机械刺激激活的整联蛋白。蛋白质活性研究发现，经刺激后，局灶性粘附激酶（FAK）以及细胞外信号调节激酶（ERK）的磷酸化状态均增加，表明这两个信号级联导致细胞周期活性的增加。需要进一步的研究来确定纤连蛋白隐蔽位点和刺激过程中激活的下游信号级联之间的联系。这些细胞行为研究都将有助于更好地了解机械环境对活组织系统的影响程度。

著录项

  • 作者

    Alger, Tara.;

  • 作者单位

    Wayne State University.;

  • 授予单位 Wayne State University.;
  • 学科 Cellular biology.
  • 学位 M.S.
  • 年度 2013
  • 页码 82 p.
  • 总页数 82
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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