逆转录聚合酶链反应（RT-PCR）：一种检测碱性成纤维细胞生长因子诱导人脐动脉内皮细胞中早期生长反应基因-1（egr-1）表达的灵敏方法。

Reverse transcriptase-polymerase chain reaction (RT-PCR): a sensitive method to examine basic fibroblast growth factor-induced expression of the early growth response gene-1 (egr-1) in human umbilical arterial endothelial cells.

作者信息

Ko Y, Totzke G, Schiermeyer B, Zeitler H, Schmitz U, Vetter H, Sachinidis A

机构信息

Medizinische Universitäts-Poliklinik, Bonn, Germany.

出版信息

Mol Cell Probes. 1995 Aug;9(4):215-22. doi: 10.1016/s0890-8508(95)90070-5.

DOI:10.1016/s0890-8508(95)90070-5

PMID:7477016

Abstract

Immediate-early genes are expressed upon growth and differentiation in a large variety of cells and species. In the present study we investigated the effect of basic fibroblast growth factor (bFGF) on early growth response gene-1 (egr-1)-mRNA expression in human umbilical arterial endothelial cells (HUAEC). The detection of this gene in HUAEC was performed by Northern blotting and by reverse transcriptase-polymerase chain reaction (RT-PCR). For RT-PCR specific primers for egr-1 and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were constructed and PCR conditions were optimized. bFGF induced a time- and concentration-dependent increase of egr-1 expression. Maximal expression occurred within 30 min of stimulation with bFGF at a concentration of 50-100 ng ml-1. RT-PCR gave highly reproducible and specific results. The comparison of both methods showed comparable results but a higher sensitivity for RT-PCR in detecting the egr-1 mRNA. RT-PCR is an excellent method for detecting the expression of egr-1 mRNA in HUAEC.

摘要

即刻早期基因在多种细胞和物种的生长与分化过程中得以表达。在本研究中，我们调查了碱性成纤维细胞生长因子（bFGF）对人脐动脉内皮细胞（HUAEC）中早期生长反应基因-1（egr-1）-mRNA表达的影响。通过Northern印迹法和逆转录-聚合酶链反应（RT-PCR）对HUAEC中的该基因进行检测。针对RT-PCR构建了egr-1和甘油醛-3-磷酸脱氢酶（GAPDH）的特异性引物，并优化了PCR条件。bFGF诱导egr-1表达呈时间和浓度依赖性增加。在用浓度为50 - 100 ng/ml的bFGF刺激30分钟内出现最大表达。RT-PCR给出了高度可重复且特异的结果。两种方法的比较显示结果具有可比性，但RT-PCR在检测egr-1 mRNA方面具有更高的灵敏度。RT-PCR是检测HUAEC中egr-1 mRNA表达的一种出色方法。