Lederer E B, Bastani K, Goebel F D
Medizinische Poliklinik, University of Munich, Germany.
Res Exp Med (Berl). 1996;196(2):77-86. doi: 10.1007/BF02576830.
Endothelin has been found to be the most powerful and important factor regulating vasoconstriction in normal and pathological conditions. Rapid degradation and very low concentrations make it difficult to use even very sensitive enzymatic or immunochemical assays for this oligopeptide. We have established a quantitative polymerase chain reaction (PCR) assay for the mRNA of endothelin-1 (ET-1), the best characterized of the three endothelin isoforms known so far. Using human umbilical vein endothelial cells as source for endothelin, the absolute number of ET-1 cDNA molecules serving as template for the amplification reaction can be calculated with the aid of an internal standard as competitor for the PCR primers, while relative amounts of ET-1 mRNA are estimated with a new solid-phase enzyme-linked immunosorbent assay for the PCR product of ET-1 and a constitutively expressed single copy gene product, beta-actin. The combination of these supplementary assay systems allows the analysis of endothelin-1 mRNA regulation on a very sensitive level.
内皮素被发现是在正常和病理条件下调节血管收缩的最强大且最重要的因素。其快速降解和极低浓度使得即便使用非常灵敏的酶法或免疫化学分析法来检测这种寡肽都很困难。我们已经建立了一种针对内皮素-1（ET-1）mRNA的定量聚合酶链反应（PCR）检测方法，ET-1是目前已知的三种内皮素异构体中特征最明确的一种。以人脐静脉内皮细胞作为内皮素的来源，借助作为PCR引物竞争物的内标，可以计算出作为扩增反应模板的ET-1 cDNA分子的绝对数量，而ET-1 mRNA的相对含量则通过一种针对ET-1的PCR产物和一种组成性表达的单拷贝基因产物β-肌动蛋白的新型固相酶联免疫吸附测定法来估算。这些补充检测系统的结合使得能够在非常灵敏的水平上分析内皮素-1 mRNA的调控情况。
