Second Harmonic Generation analysis of the ECM in idiopathic pulmonary fibrosis

 描述(由申请人提供)：特发性肺纤维化中细胞外基质的第二次谐波生成分析特发性肺纤维化(IPF)是一种以肺部结疤和僵硬为特征的疾病，在美国每年估计导致34,000人死亡。不幸的是，IPF患者的治疗选择极其有限，因为还没有找到有效的药物来阻止纤维化的进展。尽管胶原蛋白对正常和改建的ECM的结构组织都很重要，但对于IPF中的胶原结构与正常组织结构的不同之处知之甚少。显然有必要开发高度特异/敏感的技术来探测IPF组织中的胶原结构和组织。在这个项目中，我们将使用二次谐波(SHG)的高分辨率显微技术来实现新的胶原蛋白特异性分析。这种方法对纤维组织和大分子和超分子组装的亚分辨方面都很敏感。在这里，我们将利用SHG显微镜来：1)确定病理性胶原组织(在IPF中可见)与正常组织的不同之处；2)识别和量化IPF肺标本中活动性纤维化(富含III型胶原)和成熟纤维化(高I型胶原)的区域；3)评估疾病进展过程中弹性蛋白和胶原分布的变化；以及4)IPF组织标本中高III型胶原信号的区域与疾病活动的临床标志物相关。作为该项目的一部分， 我们将开发定制的自动机器视觉程序，根据严重程度自动对组织进行分类。我们将把我们所有的努力集中在研究成纤维细胞病灶周围的结构上，这将通过其他显微镜方式来识别。这些病灶被认为处于ECM重塑的前沿，但它们形成的动力学与整个纤维化过程的关系尚不清楚。我们假设这些结构变化将作为IPF的无标记生物标记物，并进一步假设胶原蛋白以一种与疾病进展相关的方式在病灶周围特异性地改变。所获得的信息可能构成未来预后/诊断方案的基础。我们提出了两个目标：目标1极化分辨倍频，以确定在IPF不同阶段COL I/III的分布和其他ECM的变化。目的2.建立肺间质纤维化SHG可视化形态变化分类系统。

 DESCRIPTION (provided by applicant): Second Harmonic Generation analysis of the ECM in idiopathic pulmonary fibrosis Idiopathic pulmonary fibrosis (IPF) is a disorder characterized by unrelenting scarring and stiffening of the lungs that leads to the death of an estimated 34,000 individuals in the U.S. each year. Unfortunately, individuals with IPF have extremely limited treatment options, as no effective drugs have been identified to halt the progression of fibrosis. Despite the importance of collagens to the structural organization both normal and remodeled ECM, little is known about how collagen structure in IPF differs from that of normal tissue architecture. There is a clear need to develop highly specific/sensitive techniques to probe collagen structure and organization in IPF tissues. In this project we will implement new collagen specific analyses using the high resolution microscopy technique of Second Harmonic Generation (SHG). This method is sensitive to both the fibrillar organization and also sub-resolution aspects of macro and supramolecular assembly. Here we will utilize SHG microscopy to: 1) determine the how pathologic collagen organization (seen in IPF) differs from normal tissue; 2) identify and quantify areas of active fibrosis (enriched in collagen III) from "old" or mature fibrosis (high in collagen I) in IPF lung specimens; 3) assess changes in elastin and collagen distribution during disease progression; and 4) correlate areas of high collagen III/I signal in IPF histologic samples with clinical markers of disease activity. As part of the project, we will develop customized automated machine vision routines to automatically classify tissues in terms of severity. We will specifically focus all of our efforts on studying structure around fibroblastic foci, which will be identified by other microscope modalities. These foci are thought to be at the leading edge of ECM remodeling but the dynamics of their formation in relationship to the overall fibrotic process remain unclear. We hypothesize that these structural changes will serve as label- free biomarkers of IPF and further hypothesize that the collagen is altered specifically around foci in a manner which is associated with disease progression. The information gained may form the basis of future prognostic/diagnostic schemes. We propose 2 Aims: Aim 1 Polarization resolved SHG to determine distribution of Col I/III and other ECM changes in different stages of IPF. Aim 2. Develop classification system of morphological changes in IPF visualized by SHG.