Biophysical Methods and Models

生物物理方法和模型

• 批准号: 8941424
• 负责人: Ralph Nossal
• 金额: $ 25.4万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8941424
• 关键词: Affect; Alcohols; Bacterial Infections; Behavior; Biocompatible Materials; Biological; Biological Assay; Cell Adhesion; Cells; Chemicals; Chemotactic Factors; Chemotaxis; Collagen; Complex; Computer Simulation; Data Analyses; Dehydration; Development; Diffuse; Diffusion; Dissociation; Drops; Drug Delivery Systems; Environment; Equation; Eukaryotic Cell; Extracellular Matrix; Fluorescence; Fourier Transform; Gel; Goals; Growth Factor; Guidelines; Human Resources; Hydrogels; Immune response; Link; Locomotion; Malignant Neoplasms; Manuscripts; Measurement; Measures; Mechanics; Medical; Methodology; Methods; Microtubules; Motion; Movement; National Cancer Institute; Neoplasm Metastasis; Optics; Peptides; Pharmaceutical Preparations; Physics; Polymers; Predisposition; Property; Reporting; Rhodamine; Sampling; Scheme; Sepharose; Signal Transduction; Site; Solutions; Spectrum Analysis; Spottings; Structure; Surface; System; Techniques; Technology; Testing; Time; Tissue Engineering; Tissues; Work; base; biophysical model; biophysical techniques; cell motility; crosslink; design; fMet-Leu-Phe receptor; falls; fluorophore; improved; interest; macromolecule; nanoprobe; neutrophil; particle; polyacrylamide; research study; response; tissue culture; tissue phantom; tissue regeneration; tissue/cell culture

The continuing goal of this multifaceted project is to develop and employ mathematical and physics-based methodologies which can be used to investigate complex biological structures and materials. One example from this reporting period is a collaborative project to provide the basis for quantitative assessment of the chemotactic response of polymorphonuclear (PMN) leukocytes and other amoeboid cells (carried out with personnel at the National Cancer Institute). Migration of cells along gradients of effector molecules, i.e., chemotaxis, is necessary during immune response and is involved in tissue development and cancer metastasis. The experimental assessment of chemotaxis thus is of high interest. The agarose spot assay is a simple tissue culture system used to detect directed cell movement induced by gradients of diverse materials, such as peptides released from the sites of bacterial infection and macromolecules that serve as growth factors. Direction sensing requires that gradients be appropriately steep, and it is necessary to know how the chemical gradients developed in this assay change over time so proper conditions for measuring chemotaxis can be effected. We have used numerical solution of the Diffusion Equation to determine the chemoattractant gradient produced in the assay. Our analysis shows that, for the usual spot size, the lifetime of the assay is optimized if the chemoattractant concentration in the spot is initially 30 times the dissociation constant of the chemoattractant-receptor bond. This result holds regardless of the properties of the chemoattractant. With this initial concentration, the chemoattractant gradient falls to the minimum threshold for directional sensing at the same time that the concentration drops to the optimal level for detecting gradient direction. This and other results of our analysis provide guidelines for application of this inexpensive and easily implemented method. A manuscript, "Improving the design of the agarose spot assay for eukaryotic cell chemotaxis," currently is under review. Related assays also are being analyzed, in conjunction with work directed at obtaining better understanding of the mechanism of cell adhesion and how it affects chemotaxis and other aspects of amoeboid locomotion. Another of our activities involved a study of the effects of pH and sample dehydration on the diffusion of small fluorescent molecules through polymer gels and solutions. This work is part of a continuing effort to understand how the properties of dense polymeric materials affect the movement of embedded molecules and nanoscopic particles. Fluorescence correlation spectroscopy (FCS) was used in this study. A unique power of FCS is that, in principle, it can detect the motions of fluorescent entities while signals from non-fluorescent surroundings can be ignored, even if the surrounding material is optically turbid (S. Zustiak, J. Riley, H. Boukari, A Gandjbakhche, and R. Nossal. J. Biomed. Optics 17:125004, 2012). For this reason, FCS increasingly is used to study particles moving in complex environments, examples being molecules moving on the surfaces of, or within, biological cells. In our recent work we used FCS to measure the translational diffusion coefficient of two fluorescent nanoprobes, rhodamine (R6G) and carboxytetramethylrhodamine (TAMRA), embedded in poly(vinyl alcohol) (PVA) solutions and gels. The diffusion coefficient was measured as a function of the pH and polymer concentration. Exchange experiments showed that the effect of pH on nanoprobe diffusion is reversible. The pH effect is observable even though the PVA chains are partially immobilized. However, interactions do not create permanent bonds between PVA and the fluorophores, as can be inferred from observations which show that R6G or TAMRA nanoprobes can diffuse freely in and out of the gels. As observed previously with other probes (A. Michelman-Ribeiro, F. Horkay, R. Nossal, and H. Boukari. Biomacromolecules 37:10212, 2004), we found that the more concentrated the solution the slower the nanoprobes move. and crosslinking the polymer chains causes the diffusion of the nanoprobes to become even slower. This behavior remains intriguing since the size of the nanoprobes (1.6 nm) is significantly smaller than that of the pores formed by the cross-linking. We also designed and built an optical chamber to determine the diffusion coefficient of PVA solutions and gels subjected to controlled dehydration, and found that dehydration induces a systematic decrease of the diffusion of TAMRA in both solutions and gels. These results demonstrate that transient physical interactions between the nanoprobes and the PVA polymers have a significant effect upon nanoprobe diffusion. Hydrogels are used in many biomedical applications, including the engineering of tissue phantoms, designing extracellular matrices for tissue regeneration, and the development of efficient drug delivery systems. Despite such important medical applications, the sieving properties of gels remain poorly understood. We are using FCS to obtain information necessary to obtain a deeper physical understanding of the complex interactions between probe particles and surrounding matrix. Similar schemes can be developed to study the movement of materials through intact tissues. Based, in part, on our understanding of gel properties, we also developed a multi-well polyacrylamide-based assay to assess how the mechanical properties of the surroundings of cells affect their fate, particlularly as relating to responsiveness to drugs. Polyacrylamide was chosen due to the ease of manipulating its stiffness and the ability to link that material to extracellular-matrix constituents. The gels were coated with collagen and used to test the effect of stiffness on the susceptibility of cancer-derived tissue culture cells to agents that target microtubules.

这个多方面的项目的持续目标是开发和采用数学和物理为基础的方法，可用于研究复杂的生物结构和材料。 本报告所述期间的一个例子是一个合作项目，该项目为定量评估多形核白细胞（PMN）白细胞和其他变形细胞的趋化反应提供了基础（与国家癌症研究所的人员一起进行）。细胞沿着效应分子梯度迁移，即，趋化性在免疫应答过程中是必需的，并参与组织发育和癌症转移。因此，对趋化性的实验评估具有很高的兴趣。琼脂糖斑点试验是一种简单的组织培养系统，用于检测由不同材料梯度诱导的定向细胞运动，例如从细菌感染部位释放的肽和用作生长因子的大分子。 方向感测要求梯度适当陡峭，并且有必要知道在该测定中产生的化学梯度如何随时间变化，以便可以实现用于测量趋化性的适当条件。我们已经使用扩散方程的数值解来确定测定中产生的化学引诱物梯度。我们的分析表明，对于通常的斑点大小，如果斑点中的化学引诱物浓度最初是化学引诱物-受体键的解离常数的30倍，则测定的寿命被优化。无论化学引诱物的性质如何，这一结果都成立。利用该初始浓度，化学引诱物梯度福尔斯下降到用于方向感测的最小阈值，同时浓度下降到用于检测梯度方向的最佳水平。我们的分析的这一结果和其他结果提供了这种廉价和易于实施的方法的应用指南。一份手稿，“改进真核细胞趋化性的琼脂糖斑点试验的设计”，目前正在审查中。 相关的分析也正在进行分析，与工作旨在获得更好的理解细胞粘附的机制，以及它如何影响趋化性和其他方面的变形虫运动。 我们的另一项活动涉及研究pH值和样品脱水对小荧光分子通过聚合物凝胶和溶液扩散的影响。这项工作是持续努力的一部分，以了解致密聚合物材料的性质如何影响嵌入分子和纳米粒子的运动。荧光相关光谱（FCS）在这项研究中使用。 FCS的一个独特功能是，原则上，它可以检测荧光实体的运动，而来自非荧光环境的信号可以被忽略，即使周围的材料是光学混浊的（S。作者：J. Boukari、A Gandjbakhche和R.诺萨J. Biomed. Optics 17：125004，2012）。因此，FCS越来越多地用于研究在复杂环境中运动的粒子，例如在生物细胞表面或内部运动的分子。在我们最近的工作中，我们使用FCS测量两种荧光纳米探针，罗丹明（R6 G）和羧基四甲基罗丹明（塔姆拉），嵌入聚乙烯醇（PVA）溶液和凝胶的平移扩散系数。 测量扩散系数作为pH和聚合物浓度的函数。 交换实验表明，pH值对纳米探针扩散的影响是可逆的。 即使PVA链被部分固定化，也可观察到pH效应。然而，相互作用不会在PVA和荧光团之间产生永久的键合，这可以从观察结果中推断出来，观察结果显示R6 G或塔姆拉纳米探针可以自由地扩散进出凝胶。正如以前用其他探针观察到的那样（A。Michelman-Ribeiro，F.霍尔凯河Nossal和H.布卡里Biomacromolecules 37：10212，2004），我们发现溶液越浓，纳米探针移动越慢。并且交联聚合物链导致纳米探针的扩散变得更慢。 这种行为仍然很有趣，因为纳米探针的尺寸（1.6 nm）明显小于交联形成的孔的尺寸。我们还设计和建造了一个光学室，以确定PVA溶液和凝胶进行控制脱水的扩散系数，并发现脱水诱导系统的塔姆拉在溶液和凝胶中的扩散减少。这些结果表明，纳米探针和PVA聚合物之间的瞬时物理相互作用对纳米探针扩散具有显著影响。水凝胶用于许多生物医学应用，包括组织模型的工程化、设计用于组织再生的细胞外基质以及开发有效的药物递送系统。 尽管如此重要的医学应用，凝胶的筛分性能仍然知之甚少。 我们正在使用FCS来获得必要的信息，以更深入地了解探针粒子和周围基质之间复杂的相互作用。 可以开发类似的方案来研究材料通过完整组织的运动。 部分基于我们对凝胶性质的理解，我们还开发了一种基于聚丙烯酰胺的多孔测定法，以评估细胞周围环境的机械性质如何影响其命运，特别是与对药物的反应性有关。 之所以选择聚丙烯酰胺，是因为它的刚度易于控制，并且能够将该材料与细胞外基质成分连接起来。用胶原蛋白包被凝胶，并用于测试刚度对癌症来源的组织培养细胞对靶向微管的试剂的敏感性的影响。