摘要
目的 建立一种敏感的检测组织型纤溶酶原激活物 ( t- PA)及其抑制剂 ( PAI- 1)基因表达的方法。 方法 用异硫氰酸胍一步法提取细胞总 RNA,用单管 RT- PCR法逆转录 -扩增 t- PA、PAI- 1和内参照三磷酸甘油醛脱氢酶 ( GAPDH) m RNA,扩增产物经电泳分离后 ,用凝胶图像分析仪照相和扫描分析 ,测定各条带分子量和光密度值。 结果 PCR产物均与预期长度相吻合 ,且PCR产物量与扩增初始模板量之间存在良好的量 -效关系。 结论 本实验建立的单管 RT- PCR半定量法是检测 t- PA、PAI- 1m RNA表达的有效手段 ,具有灵敏、快速、简便、可靠等优点。
To establish a sensitive method to examine tissue type plasminogen activator(t PA) and plasminogen activator inhibitor 1(PAI 1) mRNA expression in human umbilical vein endothelial cells(HUVECs) \ Methods\ Total cellular RNA was isolated with a single extraction with acid guanidinium thiocyanate phenol chloroform(AGPC) mixture \ A one tube coupled reverse transcription/polymerase chain reaction method(RT PCR) was used to amplify successfully t PA,PAI 1 and internal control glyceradehyde phosphate dehydrogenase(GAPDH) mRNA with respective primers \ PCR products were separated on 2% agarose gels \ With digital imaging and analysis systems, gels were photographed and scanned,then the molecular weight of each band was calculated and the integrated density value(IDV) determined \ Results\ The size of PCR products was as long as respected,and there was a dose dependent manner between the amounts of RT PCR products and RNA doses \ Conclusion\ We have established a reliable semi quantitation one tube RT PCR method to examine the expression of t PA and PAI 1 mRNA in HUVECs \;
出处
《福建医科大学学报》
1999年第3期271-275,共5页
Journal of Fujian Medical University
基金
福建省卫生厅科研基金!(970 3 0 )
关键词
脐静脉
内皮细胞
T-PA
PAI-1
mRNA
RT-PCR
reverse transcription polymerase chain reaction
gene expression
tissue type plasminogen activator genetics
plasminogen activator inhibitor 1 genetics
