Cell type-specific mRNA quantitation in non-neoplastic tissues after laser-assisted cell picking
Cell type-specific mRNA quantitation in non-neoplastic tissues after laser-assisted cell picking
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DOI：
10.1159/000055922
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发表时间：
2000-01-01
期刊：
影响因子：
5
通讯作者：
Fink, L
中科院分区：
文献类型：
--
作者：
Bohle, RM;Hartmann, E;Fink, L
目的：细胞类型特异性mRNA定量可以可靠地进行后收获不到20个细胞的轮廓从haemalaun染色的冷冻切片通过激光辅助细胞采摘，到目前为止，它一直不清楚在何种程度上这些技术可以用于分析复杂组织中的差异基因表达。研究方法：使用实验性内毒素引发的肺的大鼠模型，从气溶胶脂多糖/干扰素-γ刺激后的离体灌注和通气的大鼠肺显微解剖各种肺细胞类型。结果如下：实时RT-PCR可以定量动脉内皮细胞（AEC）、细支气管上皮细胞（BEC）、含单核细胞/巨噬细胞的肺泡隔（AS+）、无单核细胞/巨噬细胞的肺泡隔（AS-）和腔内肺泡巨噬细胞（AM）中的胆色素原脱氨酶管家基因（PBGD）和一氧化氮合酶II（NOSII）mRNA，NOSII mRNA的上调最强发生在AM中，NOS Ⅱ mRNA在BEG、AS+和AS-中表达最低，在AEC中未检测到。结论：激光显微切割和实时RT-PCR的结合是一个有价值的工具，在复杂的组织中的差异基因表达的定量原位表征。版权所有（C）2001 S. Karger AG，巴塞尔。
Objective: Cell type-specific mRNA quantitation can be reliably performed after harvesting less than 20 cell profiles from haemalaun-stained cryosections by laser-assisted cell picking, Up to now it has been unclear to what extent these techniques can be used to analyze differential gene expression in complex tissues. Methods: Using a rat model of experimental endotoxin priming of the lung various pulmonary cell types were microdissected from isolated perfused and ventilated rat lungs after aerosol lipopolysaccharide/interferon-gamma stimulation. Results: Porphobilinogen deaminase housekeeping gene (PBGD) and nitric oxide synthase II (NOSII) mRNA in arterial endothelial cells (AEC), bronchiolar epithelial cells (BEC), alveolar septum containing monocytes/macrophages (AS+), alveolar septum without monocytes/macrophages (AS-) and intraluminar alveolar macrophages (AM) could be quantified by real-time RT-PCR, The strongest upregulation of NOSII mRNA occurred in AM, while minimal NOSII expression was detected in BEG, AS+ and AS-, In AEC NOSII mRNA was not detectable. Conclusion: The combination of laser microdissection and real-time RT-PCR is a valuable tool for the quantitative in situ characterization of differential gene expression within complex tissues. Copyright (C) 2001 S. Karger AG, Basel.