Dynamic Biochemical Tissue Analysis: A Novel In Situ Assay

描述(由申请人提供)：组织学在生物医学研究、临床实践以及诊断学和治疗学的发展中无处不在。一些最强大的组织学技术是那些表征组织切片生物化学的技术。这种表征通常是通过在最大化特定识别的单一条件下分析分子探针与组织切片的结合来实现的。我们假设生化组织分析的全部能力没有实现，因为(A)当前的分析不允许探针和组织切片之间的接触模式和施加在键上的力的系统变化，以及(B)只探索了探针和组织切片之间的单一类型的相互作用，即高亲和力相互作用。我们将通过完成以下具体目标来研究这一假设：1.确定分子探针与组织切片之间的接触方法对生化组织分析分析结果的影响程度。我们开发了一种名为动态生化组织分析(DBTA)的技术，它允许系统地研究分子探针与生物组织样本的相互作用。为此，我们将最终确定探针分子与组织相互作用的条件(例如，固定在微球上的探针、施加的剪切力)是否显著影响生化组织分析的结果。确定低亲和力探针-组织切片相互作用的特征是否可以重复区分癌组织和正常组织，以及癌组织的变体。我们对DBTA的初步研究揭示了探测分子(E-选择素)与癌症组织切片之间的低亲和力相互作用。在一组条件下，这些相互作用(A)在癌组织上比正常组织更多，(B)在不同的肿瘤变种之间在统计学上是不同的。为了达到这个目的，我们将进行系统的DBTA，以确定每个癌症变异体的选择素结合谱是否独一无二，是否与正常组织不同。这些目标的完成将使我们深入了解我们假设的有效性，并将(A)提供一种新的方法来分析生物组织和原位识别抗原，(B)为理解生化组织分析中可操作的生物物理参数之间的关系提供一个框架，(C)确定可以进行DBTA以实现对各种肿瘤类型的识别的条件，(D)提出对病理组织进行分类的新方法，以及(E)为开发新的诊断学开辟新的途径。该项目将为本科生和研究生提供重要的研究机会，从而实现R15资助机制的目标。 公共卫生相关性：组织的生化分析与医学研究和临床实践密切相关，在世界各地被用来帮助识别、分析和预防疾病。这项拟议的项目寻求开发一种新的方法来分析组织切片的生物化学，该方法融合了病理学和细胞黏附领域。拟议的研究结果将对组织样本的分析产生重大影响，无论是出于研究目的还是诊断/预后目的。

DESCRIPTION (provided by applicant): Histology is ubiquitous in biomedical research, clinical practice, and the development of diagnostics and therapeutics. Some of the most powerful histological techniques are those that characterize the biochemistry of tissue sections. This characterization is typically achieved by assaying the binding of a molecular probe to a tissue section under a single condition that maximizes specific recognition. We hypothesize that the full power of biochemical tissue analysis is unrealized due to the fact that (a) current assays do not allow systematic variation of the mode of contact between the probe and the tissue section and the force exerted on the bond and (b) only a single type of interaction, a high affinity interaction, between the probe and the tissue section is explored. We will investigate this hypothesis by completing the following specific aims: I. To determine the extent to which the method of contact between the molecular probe and the tissue section influences the results of a biochemical tissue analysis assay. We have developed a technique, termed dynamic biochemical tissue analysis (DBTA), which allows systematic investigation of the interaction of molecular probes with biological tissue samples. In this aim, we will definitively determine if the conditions under which the probing molecule interacts with the tissue (e.g., probes immobilized on microspheres, applied shear force) significantly influences the results of a biochemical tissue analysis. II. To determine if characterization of low affinity probe-tissue section interactions can reproducibly discriminate between cancerous and normal tissue, and between variants of cancerous tissue. Our preliminary studies with DBTA have revealed low affinity interactions between a probing molecule (E-selectin) and cancer tissue sections. Under one set of conditions, these interactions (a) are more numerous on cancerous tissue relative to normal tissue and (b) are statistically different amongst tumor variants. In this aim we will conduct a systematic DBTA to determine if the selectin binding profile for each cancer variant is unique and different from normal tissue. Completion of these aims will give insight into the validity of our hypothesis and will also (a) provide a new approach for analyzing biological tissue and identifying antigens in situ, (b) provide a framework for understanding relationships between the biophysical parameters operative in biochemical tissue analysis, (c) identify conditions under which DBTA can be performed to achieve identification of various tumor types, (d) suggest new ways to classify pathological tissue, and (e) open new avenues for the development of novel diagnostics. The project will provide significant research opportunities for undergraduate and graduate students, thus fulfilling the objectives of the R15 grant mechanism. PUBLIC HEALTH RELEVANCE: The biochemical analysis of tissue is germane to medical research and clinical practice and is used throughout the world to aid the identification, analysis, and prevention of disease. The proposed project seeks to develop a novel approach for analyzing the biochemistry of tissue sections that merges the fields of pathology and cell adhesion. The results of the proposed study will have a significant impact on how tissue samples are analyzed both for research and diagnostic/prognostic purposes.