博来霉素诱导后细支气管上皮的激光捕获显微切割及实时逆转录/聚合酶链反应

Laser capture microdissection and real-time reverse transcriptase/ polymerase chain reaction of bronchiolar epithelium after bleomycin.

作者信息

Betsuyaku T, Griffin G L, Watson M A, Senior R M

机构信息

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

出版信息

Am J Respir Cell Mol Biol. 2001 Sep;25(3):278-84. doi: 10.1165/ajrcmb.25.3.4466.

Abstract

Terminal airways are affected in many lung diseases and toxic inhalations. To elucidate the changes in terminal airways in these diverse situations it will be helpful to profile and quantify gene expression in terminal bronchiolar epithelium. We used laser capture microdissection (LCM) to collect terminal bronchiolar epithelial cells from frozen sections of lungs of mice subjected to intratracheal bleomycin. The RNA from these cells was used for analysis of select messenger RNAs (mRNAs) by quantitative real-time polymerase chain reaction (PCR). In parallel, we used real-time PCR to analyze mRNAs in whole-lung homogenates prepared from other mice given intratracheal bleomycin. We found reductions of Clara cell-specific protein and keratinocyte growth factor receptor mRNAs in both terminal bronchiolar epithelium and whole-lung homogenates 7 d after bleomycin. In contrast, terminal bronchiolar epithelial transforming growth factor (TGF)-alpha mRNA was reduced but whole-lung TGF-alpha mRNA was not changed, whereas terminal bronchiolar epithelial epidermal growth factor (EGF) receptor mRNA was not changed but whole-lung EGF receptor was reduced. We conclude that LCM can isolate terminal bronchiolar epithelial cells for studies of cell-specific gene expression by quantitative real-time PCR, and that cell-specific gene expression in terminal bronchiolar epithelium is not necessarily reflected in analysis of whole-lung gene expression.

摘要

许多肺部疾病和有毒气体吸入都会影响终末气道。为了阐明在这些不同情况下终末气道的变化,对终末细支气管上皮中的基因表达进行分析和定量将有所帮助。我们使用激光捕获显微切割技术(LCM)从气管内注射博来霉素的小鼠肺冰冻切片中收集终末细支气管上皮细胞。这些细胞的RNA用于通过定量实时聚合酶链反应(PCR)分析选定的信使RNA(mRNA)。同时,我们使用实时PCR分析从其他气管内注射博来霉素的小鼠制备的全肺匀浆中的mRNA。我们发现博来霉素注射7天后,终末细支气管上皮和全肺匀浆中克拉拉细胞特异性蛋白和角质形成细胞生长因子受体mRNA均减少。相比之下,终末细支气管上皮转化生长因子(TGF)-α mRNA减少,但全肺TGF-α mRNA未改变,而终末细支气管上皮表皮生长因子(EGF)受体mRNA未改变,但全肺EGF受体减少。我们得出结论,LCM可分离终末细支气管上皮细胞用于通过定量实时PCR研究细胞特异性基因表达,并且终末细支气管上皮中的细胞特异性基因表达不一定反映在全肺基因表达分析中。

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