实时荧光定量RT-PCR检测内皮细胞t-PA mRNA方法的建立

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 G reen I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-PA mRNA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-PA mRNA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。

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

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.