RT-PCR法测定内皮素基因表达的建立和初步应用

Establishment of RT-PCR and Its Use in Analysis of Endothelin Gene Expression

为阐明内皮素在高血压、动脉粥样硬化及再狭窄发病过程中的作用，建立一种敏感的内皮素基因表达测定方法，该研究采用液氮冷冻组织、盐酸胍、氯仿异戊醇抽提RNA，按PCRDESN程序设计引物进行合成，将mRNA区转录为cNDA，用[α－32P]dCTP掺入PCR扩增，并对特异性条带进行β放射性计数。用反转录──多聚酶链式反应方法测出正常兔心、肺和血管组织中的内皮素mRNA表达。此方法系一种灵敏、简捷、特异的半定量测定内皮素mRNA的方法。

Endothelin (ET) is a potent vasoconstrictor and mitogenesis peptide. In order to demonstrate its pathogenic role in hypertension and arthrosclerosis. The study of the regulative mechanism of ET gene expression is necessary. It has great significance for the prevention and treatment of ET related cardiovascular disease at gene level. Tissue was minced in liquid nitrogen.RNA was isolated with guanidinium-HCl, phenol/chloroform/isoamyl alcohol. Primers were synthesized to match the sequences of rabbit ET-1 cDNA according to computer PCRDESN program.Messenger RNA was reverse transcripted into cDNA by reverse transcriptase (AMV). [α- 32P] dCTP was presented to allow quantification of PCR products by liquid scintillation in a β- counter. The result suggests that RT-PCR may assay ET- 1 mRNA in normal rabbit heart, lung and arterial tissue. Conclusion: RT - PCR is a highly sensitive, simple and specific method in semi - quantify ET-1 mRNA expression