竞争性定量RT-PCR检测妊高征患者血清对脐静脉内皮细胞TR基因表达的影响

Competitive Quantitative RT-PCR Analysis of the Expression of Thioredoxin Reductase in HUVECs Exposed on Serum from PIH

目的 建立一种能有效检测培养的人脐静脉内皮细胞 (HUVEC)中硫氧还蛋白还原酶 (TR)基因表达的方法。探讨硫氧还系统与妊高征 (PIH)发病之间的关系。方法 以RT PCR方法克隆TR基因 ,用内部缺失法构建其竞争模板 ,将竞争模板加入目的基因的逆转录扩增体系中 ,根据标准曲线得出模板中目的基因的含量。用该方法分别检测 2 2例PIH患者及 15名正常孕妇血清对体外培养的HUVEC中TR基因表达水平的影响。结果 建立了竞争性定量逆转录 聚合酶链反应 (cQRT PCR)半定量分析TR基因表达水平的方法 ;用该方法测得PIH患者及正常孕妇血清均能使HUVEC中TR基因表达显著上调 ,且两者上调幅度无明显差异。结论 PIH患者内在TR基因调节缺陷是导致患者体内抗氧化能力不足的主要因素 。

Objective The purpose of this study was to assess the regulation of thioredoxin reductase(TR) gene expression in human umbilical vein endothelial cells and investigate the relationship between TR and pathogenesis of PIH. Methods Using gene recombinant technologies, a competitor DNA fragment, known as TR gene mimic, was constructed. For construction of standard curve from which the amount of target cDNA was derived, serial dilutions of the target were co amplified with constant amount of mimic using TR gene specific primers, and the intensity of bands corresponding to the target and mimic were measured. With that method, they measured the variation of TR gene expression in HUVEC which exposed on serum collected from women with PIH ( n =22) and normal pregnancies ( n =15) respectively. Results A competitive quantitative RT PCR (cQRT PCR) method to monitor the alternation of TR gene expression levels in HUVEC was developed. The expression of TR gene were both upregulated, but there was no significant differentiation between two groups. Conclusion The intracellular regulation default plays the key role in decreasing antioxidant potential, which is not the consequence of the abnormal alteration of cytokines in PIH.