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

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）再灌注损伤后肿瘤坏死因子（TNF）-α和细胞间黏附分子（ICAM）-1水平的影响。方法实验分正常+siRNA阴性转染组、模拟缺血再灌注+siRNA阴性转染组、正常+p65 siRNA转染组和模拟缺血再灌注+p65 siRNA转染组4组。采用逆转录-聚合酶链式反应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+siRNA阴性转染组的TNF-α和ICAM-1的mRNA表达水平（分别是4.96±0.16、3.33±0.30）均高于其他3组，模拟缺血再灌注+p65 siRNA转染组的ICAM-1 mRNA表达水平（1.87±0.21）高于正常+siRNA阴性转染组（1.58±0.15）和正常+p65 siRNA转染组（1.69±0.21，均P＜0.05）。模拟缺血再灌注+siRNA阴性转染组TNF-α、ICAM-1的蛋白表达水平[分别是（329.98±12.18）μg/L、（654.74±64.79）μg/L]均高于其他3组，模拟缺血再灌注+p65 siRNA转染组TNF-α、ICAM-1的蛋白表达水平[分别是（129.65±22.42）μg/L、（185.76±11.27）μg/L]均低于正常+siRNA阴性转染组[分别是（183.50±11.77）μg/L、（280.43±13.76）μg/L，均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。%Objective To investigate the effect and regulation mechanism of mimic ischemia/reperfusion (I/R) cul-ture of human umbilical vein endothelial cells (HUVECs) on levels of tumor necrosis factor(TNF)-αand intercellular adhe-sion molecule (ICAM)-1. Methods HUVECs were randomly divided into four groups:control group (normal media cell cul-ture+control siRNA transfection), mimic I/R+control siRNA transfection group (HUVECs were transfected with control siR-NA, for 48 h ,and then received mimic ischemic media culture for 30 min followed by normal media culture for 4 h), normal culture+p65 siRNA transfection group and mimic I/R+p65 siRNA transfection group. The expression levels of TNF-αand ICAM-1 mRNA and protein were determined by real-time PCR and enzyme linked immunosorbent assay (ELISA), respec-tively. Results The mRNA and supernatant protein levels of TNF-αand ICAM-1 were significantly increased in mimic I/R culture group (4.96±0.16 and 3.33±0.30)μg/L than those of other tree groups (P<0.05). The level of ICAM-1 mRNA was significantly higher in I/R+p65 siRNA transfection group (1.87±0.21)μg/L than that of control group (1.58±0.15) μg/L and normal culture+p65 siRNA transfection group [(1.69±0.21)μg/L, P<0.05]. The levels of TNF-αand ICAM-1proteins were (329.98 ± 12.18) μg/L and (654.74 ± 64.79) μg/L in mimic I/R+control siRNA transfection group, which were significantly higher than those of other three groups (P<0.05). The levels of TNF-αand ICAM-1 proteins were (129.65±22.42)μg/L and (185.76±11.27)μg/L in mimic I/R+p65 siRNA transfection group, which were significantly lower than those of contro group [(183.50±11.77)μg/L and (280.43±13.76)μg/L, P<0.05]. Conclusion The silencing of p65 through transfection of p65 siRNA in HUVECs inhibited mimic I/R-induced mRNA and protein expressions of TNF-αand ICAM-1.