Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice

摘要: Macrophage foam cells are integral in the development of atherosclerotic lesions. Gene expression analysis lesional macrophage is complicated by cellular heterogeneity plaque and presence lesions various degrees severity. To overcome these limitations, we tested ability laser capture microdissection (LCM) real-time quantitative reverse transcription PCR to selectively analyze RNA from macrophages apolipoprotein E (apoE)-deficient mice. Proximal aortic tissue sections were immunostained for macrophagespecific CD68/macrosialin a rapid (≈15-min) protocol. Alternating each animal used isolate either entire (analogous isolation whole tissue) or LCM selection CD68-positive cells. We measured mRNA levels CD68, macrophage-specific marker, α-actin, smooth muscle cell cyclophilin A, control gene. Compared with sections, CD68 greatly enriched (33.6-fold) laser-captured macrophages. In contrast LCM-derived had undetectable α-actin. illustrate this method measure changes gene expression, injected 100 μg lipopolysaccharide i.p. into apoE-deficient mice detected increased vascular adhesion molecule-1, intercellular monocyte chemoattractant protein-1 (11.9-, 32.5-, 31.0-fold, respectively). By enriching RNA, provides powerful approach study situ regulation atherosclerosis-related genes. This will allow under conditions formation, regression, response genetic environmental perturbations.