Beer Michael, Doepping Sandra, Hildner Markus, Weber Gabriele, Grabner Rolf, Hu Desheng, Mohanta Sarajo Kumar, Srikakulapu Prasad, Weih Falk, Habenicht Andreas J R
Institute for Vascular Medicine, Friedrich Schiller University of Jena, Jena, Germany.
Methods Mol Biol. 2011;755:417-28. doi: 10.1007/978-1-61779-163-5_35.
Atherosclerosis is a transmural chronic inflammatory condition of small and large arteries that is associated with adaptive immune responses at all disease stages. However, impacts of adaptive immune reactions on clinically apparent atherosclerosis such as intima lesion (plaque) rupture, thrombosis, myocardial infarction, and aneurysm largely remain to be identified. It is increasingly recognized that leukocyte infiltrates in plaque, media, and adventitia are distinct but that their specific roles have not been defined. To map these infiltrates, we employed laser-capture microdissection (LCM) to isolate the three arterial wall laminae using apoE⁻/⁻ mouse aorta as a model. RNA from LCM-separated tissues was extracted and large-scale, whole-genome expression microarrays were prepared. We observed that the quality of the resulting gene expression maps was compromised by tissue RNA carried over from adjacent laminae during LCM. To account for these flaws, we established quality controls and algorithms to improve the predictive power of LCM-derived microarray data. Our approach creates robust transcriptome atlases of normal and atherosclerotic aorta. Assessing LCM transcriptomes for immunity-related mRNAs indicated markedly distinctive gene expression patterns in the three laminae of the atherosclerotic aorta. These mouse mRNA expression data banks can now be mined to address a wide range of questions in cardiovascular biology.
动脉粥样硬化是一种累及小动脉和大动脉的透壁性慢性炎症性疾病，在疾病的各个阶段都与适应性免疫反应相关。然而，适应性免疫反应对临床明显的动脉粥样硬化，如内膜病变（斑块）破裂、血栓形成、心肌梗死和动脉瘤的影响，在很大程度上仍有待确定。人们越来越认识到，斑块、中膜和外膜中的白细胞浸润是不同的，但它们的具体作用尚未明确。为了描绘这些浸润情况，我们以载脂蛋白E基因敲除（apoE⁻/⁻）小鼠主动脉为模型，采用激光捕获显微切割（LCM）技术分离动脉壁的三个层。提取LCM分离组织的RNA，并制备大规模全基因组表达微阵列。我们观察到，在LCM过程中，相邻层遗留的组织RNA会影响所得基因表达图谱的质量。为了解决这些缺陷，我们建立了质量控制和算法，以提高LCM衍生微阵列数据的预测能力。我们的方法创建了正常和动脉粥样硬化主动脉的强大转录组图谱。评估LCM转录组中与免疫相关的mRNA表明，动脉粥样硬化主动脉的三个层中存在明显不同的基因表达模式。现在可以挖掘这些小鼠mRNA表达数据库，以解决心血管生物学中的一系列问题。
