目的通过定量分析雌激素受体在动脉粥样硬化兔心脏和肝脏内的相对基因表达,研究雌激素受体基因mRNA的表达水平与动脉粥样硬化(AS)发生的关系。方法手术法去势新西兰雌兔,高胆固醇饮食法复制AS模型,以管家基因-βactin作为内源性校准基因,用SYBR Green I作为荧光染料,采用实时荧光定量逆转录聚合酶链式反应(real-time RT-PCR)对ERαmRNA的表达进行了分析。结果对照组(N)、模型组(C)和假手术组(SC)主动脉硬化斑块面积分别为0、(0.75±0.24)和(0.56±0.27),饮食法复制AS模型成功;ER在对照组兔心脏和肝脏内的相对基因拷贝数分别为0.46、0.49,在模型组的去势雌兔心脏和肝脏内的相对基因拷贝数分别为0.29、0.32,在假手术组兔心脏和肝脏内的相对表达量分别为0.53、0.51。结论兔心脏、肝脏内都有雌激素受体α分布;去势AS雌兔组织内雌激素受体表达量的减少可能与动脉硬化的形成有关。
Here we report the construction of sheep PrP gene standard plasmid DNA and curve using real-time RT-PCR.Total RNA was extracted from each sample and the fragments of target gene were amplified by RT-PCR.The plasmid was constructed for calibrating unknown samples.In this study,the constructed plasmid containing only the target gene was used to construct a calibration curve.The absolute standard curve method was shown to be of high linearity,sensitivity and reproducibility.The purpose of this study is to investigate the quantification of PrP mRNA expression for knowing the scrapie pathogenesis and providing powerful tool for further studies on prion diseases pathogenesis.
