Moin Kamiar, Schwartz Donald, Mullins Stefanie R, Sloane Bonnie F
Department of Pharmacology, Wayne State University, 540 E. Canfield, Detroit, MI, USA.
Methods Mol Biol. 2009;539:49-57. doi: 10.1007/978-1-60327-003-8_3.
Expression of a given protease and of the endogenous inhibitors that regulate protease activity can be readily determined at the transcript level by using whole genome microarray chips. In the case of proteases and protease inhibitors, however, determining which cells are expressing them is often critical to understanding the functional roles of the proteases. For example, in cancer many of the proteases are derived from cells that are found in the microenvironment surrounding the tumor, e.g., fibroblasts and inflammatory cells. Proteases from both fibroblasts and inflammatory cells have been implicated in malignant progression. Therefore, it is important to recognize the origin of these molecules if one is to develop effective therapies. In this regard, mouse transgenic models and xenograft models in which human tumor cells are implanted in mice are useful tools. To profile human and mouse proteases, protease inhibitors, and protease interactors, we have developed in partnership with Affymetrix a custom, single platform, dual species chip: the Hu/Mu ProtIn chip. The Hu/Mu ProtIn chip has been validated for its ability to identify human and mouse transcripts in single species specimens and to identify and distinguish between human and mouse transcripts in dual species specimens such as xenografts. In the latter specimens, the Hu/Mu ProtIn chip has enabled us to identify host (mouse) proteases that play a protective role in development of lung tumors. Here we outline a protocol for using the Hu/Mu ProtIn chip to profile proteases, protease inhibitors, and protease interactors in tissues and cells.
通过使用全基因组微阵列芯片，可以很容易地在转录水平确定特定蛋白酶以及调节蛋白酶活性的内源性抑制剂的表达情况。然而，对于蛋白酶和蛋白酶抑制剂而言，确定哪些细胞在表达它们对于理解蛋白酶的功能作用通常至关重要。例如，在癌症中，许多蛋白酶来源于肿瘤周围微环境中的细胞，如成纤维细胞和炎症细胞。来自成纤维细胞和炎症细胞的蛋白酶都与恶性进展有关。因此，如果要开发有效的治疗方法，识别这些分子的来源很重要。在这方面，小鼠转基因模型以及将人类肿瘤细胞植入小鼠体内的异种移植模型是有用的工具。为了分析人类和小鼠的蛋白酶、蛋白酶抑制剂以及蛋白酶相互作用分子，我们与Affymetrix合作开发了一种定制的、单平台、双物种芯片：Hu/Mu ProtIn芯片。Hu/Mu ProtIn芯片已被验证具有在单物种标本中识别人类和小鼠转录本，以及在异种移植等双物种标本中识别和区分人类和小鼠转录本的能力。在后者的标本中，Hu/Mu ProtIn芯片使我们能够识别在肺肿瘤发生过程中起保护作用的宿主（小鼠）蛋白酶。在这里，我们概述了一种使用Hu/Mu ProtIn芯片分析组织和细胞中的蛋白酶、蛋白酶抑制剂以及蛋白酶相互作用分子的方案。
