CAJ | 학술논문

目的观察雷帕霉素对培养的人脐静脉内皮细胞(HUVEC)抗栓功能的影响.方法将体外培养的HUVEC中加入不同浓度[0(对照组)、0.1、1和10 ng/ml]的雷帕霉素,孵育2、4、8、12和24 h后分别用实时定量RT-PCR和Western印迹方法检测组织因子(TF)、纤溶酶原激活剂抑制物(PAI-1)、内皮源性一氧化氮合酶(eNOS)和血栓调节蛋白(TM)的mRNA和蛋白表达;采用血液凝固分析仪评估HUVEC的凝血功能.结果雷帕霉素呈浓度依赖性提高了TF和PAI-1的表达,抑制了eNOS和TM表达,mRNA水平在孵育后8 h最明显,蛋白表达在12 h最为显著,与正常HUVEC(对照组)相比,eNOS的表达降低了65%(P＝0.006),TM降低了52%(P=0.005),PAI-1升高了1.7倍(P=0.009),TF升高了2.8倍(P=0.006),持续至24 h;雷帕霉素可显著缩短HUVEC的凝血时间[对照组(152±17)s与雷帕霉素组(89±9)s,P=0.005].结论雷帕霉素呈浓度依赖性升高了促栓基因TF和PAI-1表达,抑制了抗栓基因eNOS和TM表达,从而导致内皮抗栓功能失调,凝血时间缩短,这为进一步认识雷帕霉素洗脱支架置入后血栓形成的机制提供了实验依据.
목적 관찰뢰파매소대배양적인제정맥내피세포(HUVEC)항전공능적영향.방법 장체외배양적HUVEC중가입불동농도[0(대조조)、0.1、1화10 ng/ml]적뢰파매소,부육2、4、8、12화24 h후분별용실시정량RT-PCR화Western인적방법검측조직인자(TF)、섬용매원격활제억제물(PAI-1)、내피원성일양화담합매(eNOS)화혈전조절단백(TM)적mRNA화단백표체;채용혈액응고분석의평고HUVEC적응혈공능.결과 뢰파매소정농도의뢰성제고료TF화PAI-1적표체,억제료eNOS화TM표체,mRNA수평재부육후8 h최명현,단백표체재12 h최위현저,여정상HUVEC(대조조)상비,eNOS적표체강저료65%(P＝0.006),TM강저료52%(P=0.005),PAI-1승고료1.7배(P=0.009),TF승고료2.8배(P=0.006),지속지24 h;뢰파매소가현저축단HUVEC적응혈시간[대조조(152±17)s여뢰파매소조(89±9)s,P=0.005].결론 뢰파매소정농도의뢰성승고료촉전기인TF화PAI-1표체,억제료항전기인eNOS화TM표체,종이도치내피항전공능실조,응혈시간축단,저위진일보인식뢰파매소세탈지가치입후혈전형성적궤제제공료실험의거.
Objective To explore the effects of sirolimus upon endothelial thrombotic function of human umbilical vein endothelial cells(HUVEC).Methods Sirolimus was added into the in vitro cultured HUVEC at the concentrations of 0(control),0.1,1 and 10 ng/ml.At 2,4,8,12 and 24 h postincubation,the ceils were harvested for determination of tissue factor(TF),plasminogen activator inhibitor (PAI-1),endothelial nitric oxide synthase(eNOS)and thrombomodulin(TM)expression by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.The clotting functions of HUVEC were assayed by an automated coagulation analyzer.Results Sirolimus induced the expressions of TF and PAI-1 and inhibited the expressions of eNOS and TM in a concentration-dependent manner.The maximal change of mRNA expression was observed at 8 h and remained up to at least 24 h.And the most marked change of protein expression(65%reduction in eNOS expression,52%reduction in TM,1.7-fold increase in PAI-1 and 2.8-fold increase in TF)was at 12 h.The clotting time in sirolimus group(89 s±9 s)was significantly shorter than that in control group(152 s±17 s,P=0.005).Conclusion Sirolimus induces endothelial antithrombotic dysfunction and shortens the clotting time through an elevated expression of prothrombotic genes TF and PAI-1 and a lowered expression of antithrombotic genes eNOS and TM.It may be one of mechanisms of thrombosis after the implantation of sirolimus-eluting stents.