实时荧光定量RT-PCR检测内皮细胞t-PA mRNA方法的建立  

Establishment of a real-time fluorescence quantitative RT-PCR for measurement of t-PA mRNA expression in endothelial cells

作　　者：赵砚婷 张莲芬 金坚 

机构地区：[1]江南大学教育部重点实验室.细胞与分子药理研究室,江苏无锡214036

出　　处：《中国药理学通报》2006年第5期633-637,共5页Chinese Pharmacological Bulletin

摘　　要：目的建立实时RT-PCR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SYBR GreenⅠ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-PA mRNA的干预效果。结果定量方法特异性好，检测的灵敏度达10^3拷贝，线性范围为10^3～10^10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（r^2〉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。Aim 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 using 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 10^3 copies with the linear range from 10^3 to 10^10 copies.The standard curves showed high correlations （ r^2 〉 0. 990）, The intra-assay and inter-assay variation of the method was 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.

关 键 词：实时逆转录聚合酶链反应 组织型纤溶酶原激活物 人微血管内皮细胞 

分 类 号：R332.12[医药卫生—人体生理学] R340[医药卫生—基础医学]