Laser Capture For Macromolecular Analysis Of Normal Deve

随着表达的人类基因列表的扩大，一个主要的科学和医学挑战是了解驱动正常组织形态发生和实际组织中病理损伤进展的分子事件。随着PCR、微杂交阵列和突变筛查以及蛋白质组学的改进，可以分析组织活检中数百甚至数千种不同大分子的水平。由于复杂组织由多种细胞类型组成，这些细胞类型在生物化学上和物理上受到周围细胞和分子环境的影响，因此分析发育、正常功能和疾病进展中的关键基因表达模式的任务取决于从其复杂组织环境中识别和提取特定细胞。以前，我们开发了激光捕获显微切割（LCM），以提供一种基于显微镜的，用户指导的方法，以获得来自组织切片的特定显微区域的指定细胞的纯群体，用于随后的定量，多重分子分析。我们已经合作使用我们的显微切割技术，使用神经组织的微阵列杂交和DNA修复过程的空间和时间映射进行基因表达的全球分析。使用显微切割，我们继续探索与慢性炎症诱导的浆细胞瘤诱导和进展的小鼠模型中癌症进展阶段相关的关键基因表达变化以及早期癌症侵袭。在过去的一年中，我们与NCI和CIT一起开发了一种新的快速扫描技术，能够自动地，特别是将那些在复杂组织切片中免疫染色的细胞微结合到柔性热塑性带上，具有非常高的速率（每秒扫描约50，000个细胞）和分辨率（约1微米）。随着我们新的表达显微切割技术的进一步改进，我们希望大大扩展显微切割在研究中的应用，特别是在蛋白质组学和脂质组学中，这需要高分辨率靶向和高速相结合。在临床研究中发现的与特定病理病变唯一相关的大分子组可作为诊断性成像标记物用于筛查高危人群和评价对旨在预防疾病进展的治疗的反应。从长远来看，我们正在评估我们不断发展的表达显微切割技术在临床筛查和预防疾病进展的早期干预中的潜在作用。

As the list of expressed human genes expands, a major scientific and medical challenge is to understand the molecular events that drive normal tissue morphogenesis and the progression of pathologic lesions in actual tissue. With refinements in PCR, microhybridization arrays and mutation screening, and proteomics, tissue biopsies can be analyzed for levels of hundreds or even thousands distinct macromolecules. Since complex tissues consist of multiple cells types biochemically and physically affected by surrounding cells and molecular environments, the task of analyzing critical gene expression patterns in development, normal function, and disease progression depends on the identification and extraction of specific cells from their complex tissue milieu. Previously we developed laser capture microdissection (LCM) to provide a microscope-based, user-directed method to procure pure populations of specified cells from specific microscopic regions of tissue sections for subsequent quantitative, multiplex molecular analysis. We have collaborated using our microdissection technologies on global analyses of gene expression using microarray hybridization of neural tissues and in spatial and temporal mapping of DNA repair processes. Using microdissection, we continue to explore critical gene expression changes that are associated with stages of cancer progression in a mouse model of induction and progression of plasmacytomas induced by chronic inflammation and in early cancer invasion. In the last year with NCI and CIT, we have developed a new rapid scanning technology capable of automatically, specifically microbonding onto a flexible thermoplastic tape those cells that are immunostained within a complex tissue section with very high rates (~50,000 cells scanned per sec) and resolution (~1micron). With further improvements in our new expression microdissection technique, we hope to greatly expand the utility of microdissection in research studies, particularly in proteomics and lipidomics, which require the combination of high resolution targeting and high speed that it provides. Sets of macromolecules found to be uniquely associated with a defined pathological lesion in clinical studies may serve diagnostic imaging markers in screening of populations at risk and for evaluating response to therapy designed to prevent progression. In the longer term, we are evaluating the potential role of our evolving expression microdissection technology both in clinical screening and response to early intervention designed to prevent progression.