Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice
Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice
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DOI：
10.1073/pnas.042683999
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发表时间：
2002-02-19
影响因子：
11.1
通讯作者：
Fisher, EA
中科院分区：
文献类型：
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作者：
Trogan, E;Choudhury, RP;Fisher, EA
巨噬细胞泡沫细胞在动脉粥样硬化病变的发展中是不可或缺的。病变巨噬细胞泡沫细胞的基因表达分析由于动脉粥样硬化斑块的细胞异质性和不同严重程度病变的存在而变得复杂。为了克服这些限制，我们测试了激光捕获显微解剖（LCM）和实时定量反转录PCR选择性分析载脂蛋白E （apoE）缺陷小鼠病变巨噬细胞RNA的能力。采用快速（约15分钟）方法对主动脉近端组织切片进行巨噬细胞特异性CD68/macrosialin免疫染色。从每只动物的交替切片中分离RNA，或者从整个切片（类似于从整个组织中分离）中分离RNA，或者通过LCM选择cd68阳性细胞。我们测量了巨噬细胞特异性标记CD68、平滑肌细胞标记α -肌动蛋白和控制基因亲环蛋白a的mRNA水平。与全切片相比，CD68 mRNA水平在激光捕获的病变巨噬细胞中显著升高（33.6倍）。与整个切片相比，lcm衍生的RNA具有无法检测到的α -肌动蛋白水平。为了说明这种方法测量病变巨噬细胞基因表达变化的能力，我们将100杯脂多糖i.p.注射到apoe缺陷小鼠中，并在激光捕获的病变巨噬细胞中检测到血管细胞粘附分子-1、细胞间细胞粘附分子-1和单核细胞趋化蛋白-1的mRNA表达增加（分别为11.9倍、32.5倍和31.0倍）。通过选择性富集泡沫细胞RNA， LCM为研究动脉粥样硬化相关基因的原位表达和调控提供了强有力的方法。这种方法将允许研究巨噬细胞基因在各种斑块形成、消退以及对遗传和环境扰动的反应条件下的表达。
Macrophage foam cells are integral in the development of atherosclerotic lesions. Gene expression analysis of lesional macrophage foam cells is complicated by the cellular heterogeneity of atherosclerotic plaque and the presence of lesions of various degrees of severity. To overcome these limitations, we tested the ability of laser capture microdissection (LCM) and real-time quantitative reverse transcription PCR to selectively analyze RNA from lesional macrophages of apolipoprotein E (apoE)-deficient mice. Proximal aortic tissue sections were immunostained for macrophage-specific CD68/macrosialin by a rapid (approximate to 15-min) protocol. Alternating sections from each animal were used to isolate RNA either from entire sections (analogous to isolation from whole tissue) or by LCM selection of CD68-positive cells. We measured the mRNA levels of CD68, a macrophage-specific marker, alpha-actin, a smooth muscle cell marker, and cyclophilin A, a control gene. Compared with whole sections, CD68 mRNA levels were greatly enriched (33.6-fold) in the laser-captured lesional macrophages. In contrast to whole sections, LCM-derived RNA had undetectable levels of alpha-actin. To illustrate the ability of this method to measure changes in lesional macrophage gene expression, we injected 100 mug of lipopolysaccharide i.p. into apoE-deficient mice and detected in laser-captured lesional macrophages increased mRNA expression for vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, and monocyte chemoattractant protein-1 (11.9-, 32.5-, and 31.0-fold, respectively). By selectively enriching foam cell RNA, LCM provides a powerful approach to study the in situ expression and regulation of atherosclerosis-related genes. This approach will allow the study of macrophage gene expression under various conditions of plaque formation, regression, and response to genetic and environmental perturbations.