E1A激活基因阻遏子基因表达与血管外膜成纤维细胞表型转化的关系

摘要

Objective To explore possible role of cellular repressor of E1A-stimulated genes(CREG) in the process of phenotypic switching of adventitial fibroblasts(AFs). Methods Immunofluorescent staining was performed with tissue sections from mouse carotid arteries to evaluate the relationship between the expression of CREG and smooth muscle actin-α(α-SMA) in injured arteries, especially in the adventitia. Tissue block pasted culture method was used to isolate and culture AFs. RT-PCR and Western-blot were used to detect the change of CREG andα-SMA mRNA and protein expression in AFs in the presence of different concentrations of AngⅡfor 12 h/24 h or in the presence of 100 nmol/L Ang Ⅱ for different times. Results Normal mouse carotid arteries had little α-SMA expression throughout the tunica adventitia. Arteries at day 1 and day 3 post-injury exhibited significantly higher immunofluorescence of α-SMA compared with non-injured arteries. Alpha-SMA expression began to decrease on day 7 and progressively declined on day 14. In contrast, immunofluorescent staining revealed that CREG was expressed in the adventitia of normal arteries. Expression of CREG in the adventitia of injured arteries was decreased on the 1st day, reached its lowest value on the 3rd day, and increased gradually from the 7th day, and was higher compared with that in non-injured arteries on the 14th day after injury. Similarly, the expression of CREG in AFs was very high, and AngⅡremarkably decreased mRNA and protein expression levels of CREG in a dose-dependent and time-dependent manner. Conclusions The changes in CREG expression correlate with AF phenotypic modulation, and CREG down-regulation may facilitate AF phenotypic switching into myofibroblasts (MFs).%目的：探讨E1A激活基因阻遏子（CREG）基因表达与血管外膜成纤维细胞（AFs）表型转化间的关系。方法制作小鼠颈动脉损伤模型，免疫荧光染色观察血管外膜中CREG与平滑肌肌动蛋白（α-SMA）表达变化；培养小鼠原代AFs，外源性应用血管紧张素Ⅱ（AngⅡ）诱导其表型转化，RT-PCR和Western-blot检测不同作用时间及不同浓度AngⅡ刺激AFs后CREG和α-SMA mRNA和蛋白质表达。结果血管损伤后1 d、3 d血管外膜增生显著，外膜中α-SMA表达显著上调，而CREG表达显著下调；血管损伤后7 d、14 d外膜增生有所缓解，α-SMA表达水平逐渐下降，而CREG表达也有所恢复。应用AngⅡ诱导AFs发生表型转化，AFs中α-SMA mRNA和蛋白水平显著上调，而CREG mRNA和蛋白水平显著下调，并且呈时间和剂量依赖性。结论在体和离体模型均表明AFs表型转化过程中伴随CREG基因表达下调，提示CREG基因可能参与AFs的表型转化。