Laser capture microdissection and real-time reverse transcriptase/polymerase chain reaction of bronchiolar epithelium after bleomycin
Laser capture microdissection and real-time reverse transcriptase/polymerase chain reaction of bronchiolar epithelium after bleomycin
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DOI：
10.1165/ajrcmb.25.3.4466
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发表时间：
2001-09-01
影响因子：
6.4
通讯作者：
Senior, RM
中科院分区：
文献类型：
--
作者：
Betsuyaku, T;Griffin, GL;Senior, RM
许多肺部疾病和有毒吸入物都会影响终末气道。为了阐明在这些不同的情况下，在终端气道的变化，这将是有帮助的档案和定量的基因表达在终端细支气管上皮。我们使用激光捕获显微切割（LCM）收集末端细支气管上皮细胞从冷冻切片的小鼠肺内博莱霉素。来自这些细胞的RNA用于通过定量实时聚合酶链反应（PCR）分析选择的信使RNA（mRNA）。与此同时，我们使用实时PCR来分析从其他小鼠体内注射博莱霉素制备的全肺匀浆中的mRNA。我们发现，减少克拉拉细胞特异性蛋白和角质细胞生长因子受体mRNA在两个终端细支气管上皮细胞和全肺匀浆7天后博莱霉素。相反，末端细支气管上皮转化生长因子（TGF）-α mRNA减少，但全肺TGF-α mRNA没有变化，而末端细支气管上皮表皮生长因子（EGF）受体mRNA没有变化，但全肺EGF受体减少。我们的结论是，LCM可以隔离终端细支气管上皮细胞的细胞特异性基因表达的研究，通过定量实时PCR，并在终端细支气管上皮细胞特异性基因表达不一定反映在全肺基因表达的分析。
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.