Microsystems to Manipulate Fibroblast Chemotaxis

描述（由申请人提供）：在组织修复过程中，成纤维细胞趋化，即利用分布在其表面的多种生化受体沿细胞外化学梯度方向迁移。该项目提出了一种新的、创新的趋化性研究方法，通过开发一种模型系统和方法来确定施加到成纤维细胞上的细胞外配体之间的因果关系，以及随后启动趋化性驱动信号级联所需的细胞内蛋白质的重新分布。我们的假设是，促分裂原活化蛋白激酶（MAP）级联通过影响对接蛋白（生长因子受体结合物-2（GRB 2））的分布来调节成纤维细胞迁移，GRB 2与已知的化学引诱物配体血小板衍生生长因子β（PDGFB）活化的表面受体结合。目前的项目将通过实验来测试这一假设，该实验利用我们自己的透明的、单细胞的、绰号为microLane的趋化系统，在牛韧带成纤维细胞中施加PDGFB的一维浓度梯度。在我们的系统内测试的成纤维细胞的GRB 2分子将被标记，并使用与GRB 2抗体结合的生物缀合物（Q-Dots）的荧光特征进行作图。定量结果描述GRB 2再分布作为施加的PDGFB梯度的函数将开发一种新的和前所未有的方法来操纵伤口愈合过程中的成纤维细胞趋化性。这些结果将通过无血管组织修复治疗技术的临床开发以及促进伤口愈合所需的胶原蛋白支架和生物材料的研究开发来影响人类健康。

DESCRIPTION (provided by applicant): During tissue repair, fibroblast cells chemotax, i.e. migrate in the direction of extracellular chemical gradients, using the numerous biochemical receptors distributed on their surface. This project proposes a new, innovative approach to chemotaxis study by developing a model system and methodology to identify the causality between extracellular ligands imposed onto a fibroblast cell, and the consequent redistribution of intracellular proteins needed to initiate chemotaxis-driving signal cascades. It is our hypothesis that the Mitogen-Activated Protein Kinase (MAP) cascade regulates fibroblast migration by influencing the distribution of the docking protein, Growth Factor Receptor Bound-2 (GRB2), which binds to the surface receptors activated by the known chemoattractant ligand, Platelet-Derived Growth Factor Beta (PDGFB). The current project will test this hypothesis via experiments that utilize our own transparent, single-cell, chemotaxis system nicknamed the microLane, to impose one-dimensional concentration gradients of PDGFB across bovine ligament fibroblast cells. GRB2 molecules of the fibroblasts tested within our system will be labeled and mapped using the fluorescent signatures of nanocrystal Bioconjugates (Q-Dots) bound to GRB2 antibodies, apriori. Quantitative results describing GRB2 redistribution as a function of imposed PDGFB gradients will develop a new and unprecedented method to manipulate fibroblast chemotaxis during wound healing. The results will impact Human health via clinical development of therapeutic technologies in avascular tissue repair, as well as research development of collagen scaffolds and biomaterials needed to advance wound healing.