【作者】 赵砚婷； 张莲芬； 金坚；

【Author】 ZHAO Yan-ting, ZHANG Lian-fen, JIN Jian (Department of Pharmaceupical and Molecular Biotechnology, School of Biotechnology, Southern Yangtze University, Wuxi, PR China 214036)

【机构】 江南大学生物工程学院生物制药系；

【摘要】 目的建立实时逆转录聚合酶链反应,检测人微血管内皮细胞组织型纤溶酶原激活物(t-PA)mRNA的表达。方法提取人微血管内皮细胞总RNA,经RT-PCR获得靶基因(t-PA)及管家基因(β-actin)的PCR产物。纯化后,作为标准品梯度稀释,采用SYBRGreenI定量PCR检测,建立标准曲线。方法学考核参数为特异性、线性范围、精密度和重复性。分析全反式维甲酸对内皮细胞表达t-PAmRNA的干预效果。结果定量方法特异性好,检测的灵敏度达103拷贝,线性范围为103~1010拷贝,循环阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系(r2>0.990),批内变异≤3.10%,批间变异≤4.93%。1.25~20.00μmol?L-1的全反式维甲酸能明显上调内皮细胞t-PAmRNA的表达(P<0.01),且呈剂量依赖性。结论实时荧光定量RT-PCR的方法可对t-PAmRNA的表达进行准确定量,有助于溶栓药物药理学研究和新药筛选。更多还原

【Abstract】 Objective To establish a real-time fluorescence quantitative RT-PCR for detection of t-PA mRNA expression in endothelial cells. Methods The PCR products of t-PA target gene and β-actin housekeeping gene were obtained by RT-PCR after the total RNA was extracted from human microvascular endothelial cells(HMEC-1). The purified products were employed as the standards for development of t-PA mRNA real-time PCR with SYBR Green I. The stability, specificity and sensitivity of the method were evaluated as well Result The method of t-PA mRNA real-time PCR was well established, which detected as low as 103 copies with the linear range from 103 to 1010 copies. The standard curves showed high correlations(r2 > 0.990). The intra-assay and inter-assay variation of the method was blow 3.10 % and 4.93 %, respectively. The all-trans ratinoic acid (ATRA) up-regulated t-PA mRNA expression in a dose-dependent manner(1.25 ~ 20.00 μmol ?L -1 , P < 0.01) on HMEC-1 cells. Conclusion The real-time RT-PCR is reliable to quantitatively evaluate t-PA mRNA in endothelial cells. It is helpful for the pharmacology of thrombolytics and drug screening.更多还原