Laser Capture For Macromolecular Analysis Of Normal Development And Pathology

综合分子生物学需要了解大量途径的相互作用。同样，分子医学越来越依赖于复杂的大分子诊断来指导治疗选择。组织激光捕获显微解剖（LCM）的一个基本论点是，如果没有从复杂组织中分离特定的细胞群，我们将错过数千种低拷贝数表达的受调节转录因子、细胞调节剂和受体的关键控制功能。如果没有检测到这些关键效应物的变化，对组织功能和病理的综合理解将无法有效地进行。在复杂的组织中，特别是在病理变异中，如果不首先分离特定的细胞群，就很难测量每个细胞拷贝数低的大多数分子。例如，在最近与NEI的合作中，我们采用LCM方法分离视网膜拓扑闭合部位的局部（3D）细胞，并在胚胎发育的8个时间点对基因表达进行微阵列分析。这使我们能够在动物模型中识别低拷贝数转录因子，当这些转录因子被阻断时会导致关闭的丧失。这些转录因子和大约200个其他在时间和空间上共变的基因似乎可能在先天性视网膜缺损中起作用，这是一种发生在人类视网膜上的先天性发育缺陷。我们正在开发新的数学方法，能够在这些数据集中更全面地识别驱动这种局部组织发育的特定基因网络。

Integrative molecular biology requires understanding interactions of large numbers of pathways. Similarly, molecular medicine increasingly relies on complex macromolecular diagnostics to guide therapeutic choices. A fundamental argument for laser capture microdissection (LCM) of tissues is that without separation of specific cell populations from complex tissues, we will miss critical control functions of thousands of regulated transcription factors, cell regulators, and receptors that are expressed at low copy number. Without detecting changes in many of these critical effectors, the integrative understanding of tissue function and pathology will not proceed effectively. In complex tissues - particularly among pathological variations - it is exceptionally difficult to measure the majority of molecules that are at low copy number per cell without first isolating specific cell populations. For example, in a recent collaboration with the NEI, we adapted LCM method to isolate localized (3D) cells at the site of retinal topoglogical closure and performed microarray analysis of gene expression at 8 time points of embryonic development. This enabled us to identify low copy number transcriptions factors that when blocked lead to loss of closure in animal models. These transcription factors and approximately 200 other temporally and spatially covarying genes appear likely to play a role in coloboma, an inborn developmental defect of the retina occurring in humans. We are developing novel mathematical approaches capable of more comprehensively identifying within such datasets the specific networks of genes that drive such local tissue development. The LCM techniques that we started developing fourteen years ago are now widely used in molecular analysis of genetics and gene expression changes within target cells within complex tissues. However, in global proteomic and lipid studies without molecular amplification methods, the quantity of isolated cells sufficient to perform accurate characterization of less abundant species is problematic as the microscopic visualization, targeting, and isolation in laser microdissection has a maximal rate of about 1 to 20 cells per second depending on their microscopic distribution within the tissues. Recently, in collaboration with NCI and CIT, we invented and are now refining an automatic target-directed microtransfer technique based on macromolecule-specific staining of cells not requiring user visualization or microscopic targeting and capable of much higher throughput rates. This technique (patent pending) is built on our physical understanding of thermoplastic microtransfer and uses a much simpler device and transfer films than commercial laser microdissection instruments. This rapid, automated microtransfer method has improved spatial resolution (1 micron) and is consequently particularly well-suited to isolate highly dispersed, specific cell populations (e.g., specific neurons in the brain) or specific organelles (e.g., neuronal nuclei in the brain). The spatial relationships (morphology) among the specific cells in the tissue are preserved on the transfer film. In collaboration with Drs Markey, Lippincott-Schwartz, and Morgan under an NIH Directors Challenge Award, we are applying our technology to proteomics studies of subcellular organelles. As this technology becomes more robust, we will seek to integrate the microtransfer with molecular profiling of specific organelles or isolated cells within tissues, including routine proteomic and lipidomic analyses, particularly greater sensitivity to molecular species less abundant in grosser tissue samples. If microdissection and molecular analysis can be made clinically practical, the expression levels of sets of approximately 20 to 100 critical, stage-specific disease markers within a selected cell population might provide reliable diagnosis and intermediate endpoints of response to molecular therapies in individual patients. Our analysis of large gene expression and protein databases suggests that a significant fraction of all genes is expressed in any specific cell type and that the levels of gene products universally exhibit a highly skewed power-law distribution similar to those characterizing many other complex systems. We have developed mathematical models for the evolution of such distributions that predict the observed distributions of genes, protein domains, and gene expression observed in species of increasing biological complexity. In normal human physiology, homeostasis arises from a highly robust interacting network of large number of gene products in specific cell phenotypes that interact directly through direct and hormonal interactions with many other cellular phenotypes. We foresee an evolution of molecular diagnosis from one based on the qualitative or quantitative analysis of a few key biomarker macromolecules to one in which special clustering algorithms analyze complex multivariate databases. Such analyses should permit a more complete identification of highly correlated clinical cases and allow us to characterize their response to molecular therapies specifically designed to prevent progression. We are attempting to develop new approaches for better integration of our thermoplastic microtransfer methods of microdissection with downstream macromolecular analysis to permit more routine and simpler multiplex molecular diagnostics. A key feature is using the polymer matrix in which target cells are embedded for affinity purification and then for direct optical detection within the transparent polymer. Using a variety of microscopy techniques in our lab, we seek to quantitatively characterize protocols for incorporating affinity nanoparticles in the tissue and polymer matrix. In the longer term, we foresee using in situ optical labels to quantify the spatial distributions of specific molecules captured within the microtransfer and retained following simpler purification steps. Coupling the robust and simple automatic microdissection with rapid purification and detection of species might provide unique abilities to follow macromolecular changes in normal tissue development and in human pathologies. We are working on integrating the statistics of expression levels spatially and temporally correlated within specific cell populations with pathway and transcription factor databases to provide a more integrated approach to molecular physiology. With future integration of microdissection and macromolecular analysis, we believe the critical role for many less abundantly expressed genes in determining normal function and pathological changes will be more easily studied and integrated into molecular diagnostics and selection of clinical therapies.