Laser Capture For Macromolecular Analysis Of Normal Development And Pathology

激光捕获用于正常发育和病理学的大分子分析

• 批准号: 8941426
• 负责人: Robert F Bonner
• 金额: $ 11.21万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8941426
• 关键词: Algorithms; Animal Model; Antigens; Area; Benchmarking; Biological Assay; Biological Markers; Biopsy; Blood; Brain; Cancer Histology; Cardiac; Cell Nucleus; Cells; Clinical; Clinical Pathology; Collaborations; Color; Complex; Corpus striatum structure; Cytology; Cytology Histology; Data; Development; Diabetes Mellitus; Diagnostic; Disease; Disease Marker; Disease Progression; Dose; Ethylenes; Evaluation; Evolution; Film; Formalin; Gene Expression; Genes; Genetic; Goals; Heating; Image; Imagery; Immunohistochemistry; Institution; Lasers; Legal patent; Licensing; Light; Location; Macromolecular Complexes; Malignant Neoplasms; Malignant neoplasm of prostate; Measures; Medical; Messenger RNA; Methods; Microbond; Microdissection; Microscope; Microscopic; Mitochondria; Mitochondrial DNA; Molecular; Molecular Analysis; Molecular Biology; Molecular Diagnosis; Molecular Medicine; Morphology; Myopathy; National Institute of Biomedical Imaging and Bioengineering; National Institute of Child Health and Human Development; National Institute of Drug Abuse; Neurons; Operative Surgical Procedures; Oranges; Organelles; Paraffin Embedding; Parkinson Disease; Parkinsonian Disorders; Pathology; Pathway interactions; Patient Selection; Phenotype; Physics; Physiologic pulse; Polymers; Population; Property; Proteins; Proteomics; Rattus; Reporting; Research; Research Personnel; Resolution; Role; Sampling; Series; Slide; Specimen; Speed; Staging; Staining method; Stains; Stem cells; Subcellular structure; Surface; System; Techniques; Technology; Therapeutic; Tissues; Tolonium chloride; United States National Institutes of Health; Untranslated RNA; Visual; Work; Xenon; absorption; animal tissue; base; brain cell; cancer cell; cell type; cost; design; dosimetry; genome-wide; human tissue; improved; innovation; interest; knowledge base; laser capture microdissection; macromolecule; meetings; mitochondrial DNA mutation; new technology; prototype; receptor; research study; spatial relationship; transcription factor; vinyl acetate; volunteer

Integrative molecular biology requires understanding interactions of large numbers of pathways. Similarly, molecular medicine increasingly relies on complex macromolecular diagnostics to guide therapeutic choices. However, genome-wide protein and mRNA copy number distributions in each cell type generally have highly skewed Pareto distributions in which the vast majority of genes have low expression levels. Critical integrative control molecules such as transcription factors and noncoding RNAs are generally expressed at low copy number. Thus contamination by more highly expressing cell phenotypes is particularly problematic in molecular analyses of pathological tissues without first isolating specific cell populations. Laser capture microdissection (LCM) of tissues provides a robust method to separate specific cell populations from complex tissues and thus allow evaluation of thousands of regulated transcription factors, cell regulators, and receptors that are expressed at low copy number. The LCM techniques that we invented sixteen years ago are now widely used in molecular analysis of genetics and gene expression changes within target cells within complex tissues. This microscope-based microdissection, which allows high-resolution imaging of the captured material before submission for downstream multiplex molecular analysis, has a proven role in research studies. However, the requirement for visual targeting by a researcher using specialized microscopes has limited its applications in proteomic research where an impractical number of target decisions are required for analysis of less abundant proteins. Similarly microdissection use in clinical diagnostics has been limited by subjectivity, low throughput and cost associated with targeting of specific cells in a specialized LCM microscope. We invented and patented an automatic target-activated microdissection technique based on localized absorption within a uniformly illuminated region by specifically stained cells or organelles. The specific staining determines the location of thermoplastic bond formation and the precision with which targets are automatically captured. This does not require user visualization or microscopic targeting. This technique combines our physical understanding of light-activated thermoplastic microbonding with standard histochemical staining techniques that provide high absorptive contrast for innumerable specific targets within tissues. For example, standard immunohistochemistry creates absorption specific to the presence of a specific antigen or protein. Our flashlamp target-expression activated prototypes irradiate the entire immunostained tissue section, but only the stained targets are heated and bonded to the thermoplastic polymer on its surface. The light dose needed is much less than needed in commercial laser microdissection. This reduces the thermal transients in the tissue and film and improves spatial resolution to better than 1 micron while dramatically increasing the rate at which a whole section can be precisely microdissected. Consequently the new method is particularly well suited to isolate highly dispersed, specific cell populations (e.g., specific neurons within a brain section) or specific organelles (e.g., nuclei of invasive cancer cells). The spatial relationships (morphology) among the specific cells in the tissue are preserved on the transfer film. Over the last three years, we (CIT, NIBIB, NICHD) have designed and built a series of Xenon flashlamp fTAM prototype systems that simultaneously captures all specifically stained targets within a large region of interest with 200-microsec white light pulses. The robustness and precision of this approach relies the properties of specially selected commercial EVA tapes or unique transfer films that we have developed with Nicole Morgan (NIBIB) which have an ultrathin (1 micron) thermoplastic polymer coating on a thermally stable, clear polymer film. The broad, flashtube spectrum allows spectral filtering to be optimized for the specific stain colors. For example, we have used the standard histochemical stain, toluidine blue, with orange filtered flash to capture cancer cell nuclei from a high-grade prostate cancer histology slide. We are optimizing this approach for specifically stained dopaminergic axonal terminations (1micron) within rat brain sections. We plan to integrate our ability to microtransfer such specific microstructures within the brain for a variety of multiplex molecular analyses and thereby increase our sensitivity to molecular species associated with specific subcellular structures. In our initial study we are seeking to demonstrate that we can reliably target the dopaminergic axonal terminations in order to study mitochondrial DNA changes in Parkinsonian animal models. For a number of years clinical molecular diagnostics have focused on discovery of concise sets of pathology specific biomakers that might be quantitatively assayed from a routinely accessible clinical sample (e.g., blood, biopsy or surgical sample). Microdissection currently has an important role in such discovery, by isolating phenotypically pure pathological samples. The utility of microdissection in clinical diagnostics will require integration with current clinical pathology methods while efficiently providing increased accuracy by analyzing many stage-specific disease markers within such purified phenotypic targets compared to the complex and variable mixtures of cell types in the original clinical specimen. For this to be practical, microdissection of clinical samples (e.g., formalin-fixed paraffin-embedded tissue sections and cytology specimens) must be reduced to a simpler, lower-cost, robust method. To meet this need, we are developing simple, robust approaches for integration of our thermoplastic microtransfer methods of microdissection with upstream pathology work-flow and downstream macromolecular analysis for clinically practical multiplex molecular diagnostics. Given the potential of such integration to more reliably measure larger numbers of interacting biomolecules with specific pathophysiological cells, we foresee an evolution of molecular diagnosis from one based on the qualitative or quantitative analysis of a few abundant, specific biomarker macromolecules to one in which special knowledge-based clustering algorithms characterize disease state with high-dimensional molecular data from microdissected samples. In summary, we are developing new technologies that integrate microdissection with macromolecular analysis of histology and cytology samples that would allow the study of many less abundantly expressed biomolecules in determining normal function and pathological changes. Our goal is to have our new technology commercialized and integrated into better molecular diagnostics to guide selection of patient appropriate clinical therapies.

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复杂的大分子诊断来指导治疗选择。然而，每种细胞类型中的全基因组蛋白质和 mRNA 拷贝数分布通常具有高度倾斜的帕累托分布，其中绝大多数基因具有低表达水平。转录因子和非编码RNA等关键整合控制分子通常以低拷贝数表达。因此，在不首先分离特定细胞群的情况下，在病理组织的分子分析中，更高表达的细胞表型的污染尤其成问题。组织的激光捕获显微切割 (LCM) 提供了一种从复杂组织中分离特定细胞群的稳健方法，从而可以评估数千种低拷贝数表达的受调节转录因子、细胞调节因子和受体。 我们十六年前发明的 LCM 技术现已广泛应用于复杂组织内靶细胞内的遗传学和基因表达变化的分子分析。这种基于显微镜的显微切割可以在提交下游多重分子分析之前对捕获的材料进行高分辨率成像，在研究中具有已被证明的作用。然而，研究人员使用专用显微镜进行视觉靶向的要求限制了其在蛋白质组学研究中的应用，在蛋白质组学研究中，分析不太丰富的蛋白质需要不切实际的目标决策数量。同样，显微解剖在临床诊断中的使用也受到主观性、低通量和与在专门的 LCM 显微镜中靶向特定细胞相关的成本的限制。我们发明了一种自动靶标激活显微切割技术并获得了专利，该技术基于特定染色的细胞或细胞器在均匀照明区域内的局部吸收。特定的染色决定了热塑性粘合形成的位置以及自动捕获目标的精度。这不需要用户可视化或微观目标。该技术将我们对光激活热塑性微粘合的物理理解与标准组织化学染色技术相结合，为组织内无数特定目标提供高吸收对比度。例如，标准免疫组织化学产生针对特定抗原或蛋白质的存在的特异性吸收。我们的闪光灯目标表达激活原型照射整个免疫染色的组织切片，但只有染色的目标被加热并粘合到其表面的热塑性聚合物上。所需的光剂量远低于商业激光显微切割所需的光剂量。这减少了组织和胶片中的热瞬变，并将空间分辨率提高到优于 1 微米，同时显着提高了整个切片的精确显微解剖速度。因此，新方法特别适合分离高度分散的特定细胞群（例如，大脑部分内的特定神经元）或特定细胞器（例如，侵袭性癌细胞的细胞核）。组织中特定细胞之间的空间关系（形态）保留在转移膜上。 在过去的三年中，我们（CIT、NIBIB、NICHD）设计并构建了一系列氙气闪光灯 fTAM 原型系统，该系统可以使用 200 微秒白光脉冲同时捕获大范围感兴趣区域内的所有特定染色目标。这种方法的稳健性和精确性依赖于我们与 Nicole Morgan (NIBIB) 共同开发的专门挑选的商用 EVA 胶带或独特的转移膜的特性，它们在热稳定的透明聚合物膜上具有超薄（1 微米）热塑性聚合物涂层。广泛的闪光管光谱允许针对特定染色颜色优化光谱过滤。例如，我们使用标准组织化学染色剂甲苯胺蓝和橙色过滤闪光从高级前列腺癌组织学载玻片中捕获癌细胞核。 我们正在优化这种方法，用于大鼠脑切片内特定染色的多巴胺能轴突末端（1微米）。我们计划整合我们在大脑内微转移此类特定微观结构的能力，以进行各种多重分子分析，从而提高我们对与特定亚细胞结构相关的分子种类的敏感性。 在我们的初步研究中，我们试图证明我们可以可靠地靶向多巴胺能轴突末端，以便研究帕金森病动物模型中线粒体 DNA 的变化。 多年来，临床分子诊断一直致力于发现简明的病理学特异性生物标记物，这些生物标记物可以从常规可获取的临床样本（例如血液、活检或手术样本）中进行定量分析。显微解剖目前通过分离表型纯病理样本在此类发现中发挥着重要作用。显微解剖在临床诊断中的应用需要与当前的临床病理学方法相结合，同时通过分析这种纯化表型目标中的许多阶段特异性疾病标记物（与原始临床标本中复杂多变的细胞类型混合物相比）来有效地提高准确性。为了使其实用化，必须将临床样本（例如福尔马林固定石蜡包埋的组织切片和细胞学标本）的显微切割简化为更简单、成本更低、稳健的方法。为了满足这一需求，我们正在开发简单、稳健的方法，将我们的显微切割热塑性微转移方法与上游病理工作流程和下游大分子分析相集成，以实现临床实用的多重分子诊断。鉴于这种集成有可能更可靠地测量大量与特定病理生理细胞相互作用的生物分子，我们预见分子诊断将从基于对一些丰富的特定生物标志物大分子的定性或定量分析的诊断演变为基于特殊知识的聚类算法利用来自显微解剖样本的高维分子数据来表征疾病状态的诊断。 总之，我们正在开发将显微切割与组织学和细胞学样本的大分子分析相结合的新技术，这将允许研究许多表达量较低的生物分子，以确定正常功能和病理变化。我们的目标是将我们的新技术商业化并整合到更好的分子诊断中，以指导选择适合患者的临床疗法。

项目成果

Nonlinear gene cluster analysis with labeling for microarray gene expression data in organ development.

器官发育中微阵列基因表达数据的非线性基因聚类分析和标记。

• DOI: 10.1186/1753-6561-5-s2-s3
• 发表时间: 2011
• 期刊: BMC proceedings
• 作者: Ehler,Martin;Rajapakse,VinodhN;Zeeberg,BarryR;Brooks,BrianP;Brown,Jacob;Czaja,Wojciech;Bonner,RobertF
• 通讯作者: Bonner,RobertF