Veremeyko Tatiana, Starossom Sarah-Christine, Weiner Howard L, Ponomarev Eugene D
Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School.
J Vis Exp. 2012 Jul 23(65):4097. doi: 10.3791/4097.
Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)(1). These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)(2). Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers(3). The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation(4) and pathologies such as inflammation(5). MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages(6) and microglia(7). In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.
小胶质细胞是存在于中枢神经系统（CNS）中的髓系谱系细胞（1）。这些细胞在许多与神经炎症相关的疾病（如多发性硬化症（MS））的病理过程中发挥重要作用（2）。正常CNS中的小胶质细胞表达巨噬细胞标志物CD11b，并通过表达低水平的激活标志物（如CD45）表现出静息表型。在CNS的病理事件中，小胶质细胞会因CD45和其他标志物的上调而被激活（3）。影响CNS中小胶质细胞表型和功能的因素尚未得到充分研究。微小RNA（miRNA）是一个不断增加的保守分子家族（约22个核苷酸长），参与许多正常生理过程，如细胞生长和分化（4）以及炎症等病理过程（5）。miRNA通过与其mRNA的互补序列结合来下调某些靶基因的表达，并在包括巨噬细胞（6）和小胶质细胞（7）在内的先天免疫细胞的激活中发挥重要作用。为了研究定义小胶质细胞表型、生物学功能并将小胶质细胞与其他类型巨噬细胞区分开来的miRNA介导的途径，定量评估中枢神经系统驻留小胶质细胞不同亚群中特定miRNA的表达非常重要。测量CNS中miRNA表达的常用方法包括从整个神经组织进行定量PCR和原位杂交。然而，从整个组织匀浆进行定量PCR无法评估小胶质细胞中miRNA的表达，小胶质细胞仅占神经组织细胞的5-15%。原位杂交可以评估组织切片中特定细胞类型中miRNA的表达，但该方法并非完全定量。在本报告中，我们描述了一种定量且灵敏的方法，用于通过实时PCR检测从小鼠实验性自身免疫性脑脊髓炎（EAE，一种MS小鼠模型）分离的正常CNS或神经炎症期间小胶质细胞中的miRNA。所描述的方法将有助于测量正常CNS或与包括MS、中风、创伤性损伤、阿尔茨海默病和脑肿瘤在内的各种病理相关的神经炎症期间小胶质细胞中miRNA的表达水平。
