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小干扰RNA沉默p65基因对人脐静脉内皮细胞再灌注后促炎因子表达的影响

曾敏,吴智勇,郑茵,何扬利,费毅,刘肖君   

  1. 海南省人民医院
  • 收稿日期:2013-07-08 修回日期:2013-10-26 出版日期:2014-02-15 发布日期:2014-02-15
  • 通讯作者: 曾敏

  • Received:2013-07-08 Revised:2013-10-26 Published:2014-02-15 Online:2014-02-15

摘要: 目的 通过小干扰RNA (siRNA) 研究沉默p65对人脐静脉内皮细胞(HUVECs)再灌注损伤后促炎因子表达水平的影响。方法 分为4组对HUVECs进行培养,即正常培养+siRNA阴性对照转染组、缺血再灌注+siRNA阴性对照转染组、正常培养+p65 siRNA转染组和缺血再灌注+p65 siRNA转染组。模拟缺血再灌注通过在模拟缺血培养液中培养HUVECs 30分钟后换正常培养液再培养4小时而实现,siRNA转染则在模拟缺血再灌注培养前48小时对HUVECs转染p65 siRNA或阴性对照siRNA。实时定量PCR和酶联免疫吸附法分别检测p65 siRNA对TNF-α、ICAM-1 mRNA和蛋白表达水平的影响。 结果 模拟缺血再灌注刺激TNF-α、ICAM-1的mRNA和蛋白表达水平显著升高( P < 0.05) 。p65 siRNA转染显著抑制模拟缺血再灌注所诱导的HUVECs的TNF-α、ICAM-1 的mRNA和蛋白表达水平( P < 0.05)。结论 p65 siRNA通过沉默p65,抑制模拟缺血再灌注诱导的TNF-α和ICAM-1表达水平。

关键词: 小干扰RNA, 模拟缺血再灌注, 人脐静脉内皮细胞, 促炎因子

Abstract: Objective To investigate the effect and regulation mechanism of mimic ischemia/reperfusion culture of human umbilical vein endothelial cells (HUVECs) on pro-inflammatory factors. Methods HUVECs were divided into 4 groups: control group(culture with normal media), mimic ischemia/reperfusion group(mimic ischemic media cultured for 30min followed by normal media cultured for 4h), normal culture +p65 siRNA group(HUVECs were transfected with p65 siRNA 48h before mimic ischemia/reperfusion), mimic ischemia/reperfusion group +p65 siRNA group. The mRNA and protein expressions of TNF-αand ICAM-1 in each group were determined by real-time PCR and enzyme linked immunosorbent assay (ELISA), respectively. Results The mRNA and protein levels of TNF-αand ICAM-1 were significantly increased by mimic ischemia/reperfusion (P < 0.05), which were otherwise significantly inhibited by the transfection of p65 siRNA(P < 0.05). Conclusions Silencing p65 through the transfection of p65 siRNA in HUVECs inhibited mimic ischemic/reperfusion- induced expression of TNF-αand ICAM-1.

Key words: siRNA, mimic ischemic/reperfusion, human umbilical vein endothelial cells, pro-inflammatory factors