Zhang Daohai, Koay Evelyn Siew-Chuan
Molecular Diagnosis Center, Department of Laboratory Medicine, National University Hospital, Singapore.
Methods Mol Biol. 2008;428:77-91. doi: 10.1007/978-1-59745-117-8_5.
Laser capture microdissection (LCM) is a powerful tool for procuring near-pure populations of targeted cell types from specific microscopic regions of tissue sections, by overcoming problems due to tissue heterogeneity and minimizing intermixture and contamination by other cell types. The combination of LCM with various proteomic technologies has enabled high-throughput molecular analysis of human tumors, and provided critical tools in the search for novel disease markers and therapeutic targets. As an example, we describe the application of LCM in dissecting the tumor cells in breast cancer for macromolecular extraction and subsequent protein separation by 2-dimensional gel electrophoresis (2-D GE). The protocols and the key issues involved in preparing ethanol-fixed paraffin-embedded tissue blocks and microscopic sections, microdissecting the cells of interest using the PixCell II LCM system, extracting and separating the cellular proteins by 2-D GE, and preparing selective proteins for peptide mass analysis by mass spectrometry, are discussed. The aim is to provide a practical guide in performing high-throughput microdissection of target cells and gel-based proteomics, which can be adapted to research in cancer formation and growth.
激光捕获显微切割（LCM）是一种强大的工具，可通过克服组织异质性问题并最大限度减少其他细胞类型的混合与污染，从组织切片的特定微观区域获取近乎纯净的目标细胞群体。LCM与各种蛋白质组学技术的结合，使得对人类肿瘤进行高通量分子分析成为可能，并为寻找新型疾病标志物和治疗靶点提供了关键工具。例如，我们描述了LCM在解剖乳腺癌肿瘤细胞以进行大分子提取及随后通过二维凝胶电泳（2-D GE）进行蛋白质分离中的应用。讨论了制备乙醇固定石蜡包埋组织块和显微切片、使用PixCell II LCM系统显微切割感兴趣的细胞、通过2-D GE提取和分离细胞蛋白质以及制备用于质谱肽质量分析的选择性蛋白质所涉及的方案和关键问题。目的是提供一份在进行目标细胞的高通量显微切割和基于凝胶的蛋白质组学时的实用指南，该指南可适用于癌症形成和生长的研究。
