LASER CAPTURE FOR MACROMOLECULAR ANALYSIS OF NORMAL DEVELOPMENT AND PATHOLOGY

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的改进，可以从组织活检中提取DNA或mRNA，并使用数百甚至数千个遗传标记的平行组进行分析。由于复杂组织中的细胞受到周围细胞和来自更远距离的远程刺激的生物化学和物理影响，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提取特定细胞。激光捕获显微切割（LCM）是本实验室与NCI和ORS合作开发的一种快速、可靠的方法，可以从组织切片的特定显微区域获得特定细胞的纯群体，用于随后的定量、多重分子分析。在过去的一年中，我们进一步完善了LCM仪器，用于单细胞的免疫组化靶向，并用于蛋白质和mRNA定量。通过与Arcturus Engineering，Inc（山景，CA）的合作研究与开发协议（CRADA），该公司已将这一精炼技术商业化，并提供给全球300多个研究实验室。基于LCM的组织病理学病变的分子分析可以应用于通过组织采样可访问的任何疾病过程。LCM正被用于癌症基因组解剖计划（CGAP）的努力，以目录的基因，表达在人体组织中的正常细胞经历癌前病变，并进一步发展成侵袭性和转移性癌症。与特定疾病阶段相关的表达基因的波动或细胞DNA中的改变可以在个体患者内或个体患者之间进行比较。这种基因表达模式的指纹可以为病因学提供关键线索，并最终有助于诊断决策和针对个体患者的治疗。我们的实验室正在开发强大的统计工具，以应用于这样的多维数据集，以确定关键基因，途径，以及大量基因的转录，翻译和翻译后修饰的复杂整合，这些基因表征正常细胞功能和特定病理学的偏差特征。发现与确定的病理性病变唯一相关的大分子可以作为成像或治疗靶点。我们目前正在使用这些方法研究与正常发育相关的基因表达模式。- 激光捕获显微切割，大分子分析，分子诊断，基因表达，病理学，发展

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, DNA or mRNA can be extracted from tissue biopsies and analyzed with a parallel panel of hundreds or even thousands of genetic markers. Because cells in complex tissue are biochemically and physically affected by surrounding cells and by remote stimuli from greater distances, the task of analyzing critical gene expression patterns in development, normal function, and disease progression depends on the extraction of specific cells from their complex tissue milieu. Laser capture microdissection (LCM) has been developed by our lab in collaboration with NCI and ORS to provide a rapid, reliable method to procure pure populations of specified cells from specific microscopic regions of tissue sections for subsequent quantitative, multiplex molecular analysis.In the last year we have further refined LCM instrumentation for immunohistochemical targeting of single cells and protocols for its use for protein and mRNA quantitation. Through a Collaborative Research and Development Agreement (CRADA) partnership with Arcturus Engineering, Inc (Mountain View, CA), have made this refined technology commercially available to over 300 research labs around the world. LCM-based molecular analysis of histopathological lesions can be applied to any disease process that is accessible through tissue sampling. LCM is being used in the Cancer Genome Anatomy Program (CGAP) effort to catalog the genes which are expressed in human tissue as normal cells undergo premalignant changes and further develop into invasive and metastatic cancer. The fluctuation of expressed genes or alterations in the cellular DNA which correlate with a particular disease stage can be compared within or among individual patients. Such a fingerprint of gene expression patterns may provide crucial clues for etiology, and may ultimately contribute to diagnostic decisions and therapies tailored to the individual patient. Our lab is developing robust statistical tools to be applied to such multidimensional datasets in order to identify critical genes, pathways, and the complex integration of transcription, translation, and post-translational modification of large number of genes that characterize normal cellular function and the deviations characteristic of specific pathologies. Macromolecules found to be uniquely associated with a defined pathological lesion may serve as imaging or therapeutic targets. We are currently studying gene expression patterns associated with normal development using these methods. - Laser capture microdissection, Macromolecular analysis, molecular diagnosis, gene expression, pathology, development