【摘要】 目的 构建早期生长反应因子3（Egr3）基因敲除动脉粥样硬化小鼠模型，探讨Egr3对主动脉组织炎症及细胞凋亡的影响。方法 SPF级雄性Egr3^fl/fl和Egr3^{（fl/fl, Cdh5-cre）}小鼠各16只，体质量16.2～23.5 g,将Egr3^fl/fl小鼠分为对照组（常规饲料喂养12周）、模型组（注射PCSK9腺相关病毒载体AAV8-PCSK9并高脂饲料喂养12周构建动脉粥样硬化模型）各8只，将Egr3^{（fl/fl, Cdh5-cre）}小鼠分为Egr3^-/-组（他莫昔芬注射诱导Egr3基因敲除并常规饲料喂养12周）、Egr3^-/-+模型组（他莫昔芬注射诱导Egr3基因敲除后注射AAV8-PCSK9并高脂饲料喂养12周构建动脉粥样硬化模型）各8只。4组小鼠饲料喂养期间每周称体质量，造模2、4、8、12周时采用生化试剂盒检测血糖、总胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）水平。造模12周时采用HE染色观察主动脉组织病理情况，采用油红O染色观察主动脉斑块形成情况并计算主动脉斑块面积百分比，采用TUNEL法检测主动脉组织细胞凋亡情况，采用ELISA法检测血清白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）水平，采用Western blot法检测主动脉组织Egr3、Bcl-2相关X蛋白（Bax）、B细胞淋巴瘤-2（Bcl-2）蛋白相对表达量。结果 随造模时间延长，4组体质量有逐渐增高趋势。造模2、4、8周时对照组、Egr3^-/-组血糖、TC、TG、LDL-C、HDL-C水平均无明显变化；模型组、Egr3^-/-+模型组血糖、TC、TG、LDL-C水平均逐渐升高，HDL-C水平均逐渐降低。造模12周时4组体质量、血糖、TC、TG、LDL-C、HDL-C水平比较差异均有统计学意义（F=5.867～185.639,P均<0.05）。模型组、Egr3^-/-+模型组体质量均高于对照组（P<0.05）,Egr3^-/-+模型组高于Egr3^-/-组（P<0.05）;模型组、Egr3^-/-+模型组血糖、TC、TG、LDL-C水平均高于对照组、Egr3^-/-组（P<0.05）,HDL-C水平均低于对照组、Egr3^-/-组（P<0.05）;模型组血糖、TC、TG、LDL-C水平均高于Egr3^-/-+模型组（P<0.05）。对照组主动脉管腔结构较完整，管壁厚度不均，部分内膜细胞肿胀脱落，中膜少量脂质沉积；模型组主动脉结构紊乱，部分弹性纤维肿胀，泡沫细胞增多，内膜增厚形成纤维帽；Egr3^-/-组主动脉管腔结构较完整，管壁厚度较均匀，中膜脂质沉积较对照组减少；Egr3^-/-+模型组主动脉结构较完整，内膜无明显纤维斑块沉积，中膜脂质沉积及泡沫细胞较模型组减少。Egr3^-/-组、对照组、Egr3^-/-+模型组、模型组主动脉斑块面积百分比[（1.13±0.30）%、（4.38±1.12）%、（7.64±0.34）%、（9.32±1.39）%]逐渐增大（F=61.662,P<0.001）,主动脉组织细胞凋亡率[（2.28±0.45）%、（29.35±4.94）%、（36.83±5.33）%、（57.10±3.48）%]逐渐升高（F=141.271,P<0.001）。4组血清IL-1β、TNF-α水平及主动脉组织Egr3、Bax、Bcl-2蛋白相对表达量比较差异均有统计学意义（F=13.927～639.612,P均<0.05）。模型组、Egr3^-/-+模型组血清IL-1β、TNF-α水平均高于对照组、Egr3^-/-组（P<0.05）,模型组均高于Egr3^-/-+模型组（P<0.05）。Egr3^-/-组、Egr3^-/-+模型组、对照组、模型组主动脉组织Egr3蛋白相对表达量逐渐升高（P<0.05）;模型组、Egr3^-/-+模型组主动脉组织Bax蛋白相对表达量均高于对照组、Egr3^-/-组（P<0.05）,Bcl-2蛋白相对表达量均低于对照组、Egr3^-/-组（P<0.05）;模型组主动脉组织Bax蛋白相对表达量高于Egr3^-/-+模型组（P<0.05）,Bcl-2蛋白相对表达量低于Egr3^-/-+模型组（P<0.05）。结论 Egr3在动脉粥样硬化小鼠主动脉组织中表达上调，敲除Egr3基因可减轻动脉粥样硬化病变，可能与抑制主动脉组织炎症和细胞凋亡有关。更多还原

【Abstract】 Objective To construct the early growth response factor 3（Egr3） gene knockout mice models of atherosclerosis, and to explore the effect of Egr3 on inflammation and apoptosis in aortic tissues. Methods Sixteen SPF-grade male Egr3^fl/fl mice and 16 Egr3^{（fl/fl, Cdh5-cre）} mice weighing 16.2-23.5 g were enrolled. The Egr3^fl/fl mice were equally divided into the control group（fed with regular diet for 12 weeks） and the model group（injected with PCSK9 adeno-associated virus vector AAV8-PCSK9 and fed with high-fat diet for 12 weeks to induce atherosclerosis model）, and the Egr3^{（fl/fl, Cdh5-cre）} mice were equally divided into the Egr3^-/- group（induced by tamoxifen injection for Egr3 gene knockout and fed with regular diet for 12 weeks） and the Egr3^-/-+model group（induced by tamoxifen injection for Egr3 gene knockout, followed by injection of AAV8-PCSK9 and fed with high-fat diet for 12 weeks to induce atherosclerosis model）. The body mass was measured weekly during the feeding period in these four groups. At 2, 4, 8 and 12 weeks of modeling, biochemical kits were used to detect the levels of blood glucose, total cholesterol（TC）, triacylglycerol（TG）, low-density lipoprotein cholesterol（LDL-C）, and high-density lipoprotein cholesterol（HDL-C）. At 12 weeks of modeling, HE staining was used to observe the histopathology in aortic tissues, oil red O staining was used to observe the aortic plaque formation and calculate the aortic plaque area percentage, TUNEL assay was used to detect the apoptosis in aortic tissues, ELISA was used to detect serum levels of interleukin-1β（IL-1β） and tumor necrosis factor-α（TNF-α）, and Western blot was used to detect the relative expressions of Egr3, Bcl-2 associated X protein（Bax） and B-cell lymphoma-2（Bcl-2） proteins in aortic tissues. Results As the modeling time prolonged, there was a gradual increasing trend in body mass in the four groups. At 2, 4 and 8 weeks of modeling, the blood glucose, TC, TG, LDL-C and HDL-C levels showed no significant changes in the control group and Egr3^-/- group; the blood glucose, TC, TG and LDL-C levels gradually increased, while the HDL-C level decreased gradually in the model group and Egr3^-/- + model group. At 12 weeks of modeling, there were significant differences in the body mass, blood glucose, TC, TG, LDL-C and HDL-C levels among four groups（F=5.867-185.639, all P values <0.05）. The body mass was higher in the model group and Egr3^-/-+model group than that in the control group（P<0.05）, was higher in the Egr3^-/-+model group than that in the Egr3^-/- group（P<0.05）. The blood glucose, TC, TG and LDL-C levels were higher in the model group and Egr3^-/-+model group than those in the control group and Egr3^-/- group（P<0.05）, were higher in the model group than those in the Egr3^-/-+model group（P<0.05）. The HDL-C level was lower in the model group and Egr3^-/-+model group than that in the control group and Egr3^-/- group（P<0.05）. In the control group, the aortic lumen structure was relatively intact, with uneven thickness of the aortic wall, partial intimal cells swelling and shedding, and a small amount of lipid deposition in the media. In the model group, the aortic structure was disordered, with partial elastic fibers swelling, increased foam cells, and intima thickening forming a fibrous cap. In the Egr3^-/- group, the aortic lumen structure was relatively intact, with even thickness of the aortic wall, and less lipid deposition in the media compared to the control group. In the Egr3^-/-+model group, the aortic structure was relatively intact, with no obvious fibrous plaque deposition in the intima, and less lipid deposition in the media and fewer foam cells compared to the model group. The aortic plaque area percentage increased gradually in the Egr3^-/- group, control group, Egr3^-/-+model group and model group [（1.13±0.30）%,（4.38±1.12）%,（7.64±0.34）%,（9.32±1.39）%]（F=61.662, P<0.001）, and the apoptosis rate of aortic tissues also increased gradually [（2.28±0.45）%,（29.35±4.94）%,（36.83±5.33）%,（57.10±3.48）%]（F=141.271, P<0.001）. There were significant differences in the serum levels of IL-1β and TNF-α as well as the relative expressions of Egr3, Bax and Bcl-2 proteins among four groups（F=13.927-639.612, all P values <0.05）. The serum levels of IL-1β and TNF-α were higher in the model group and Egr3^-/-+model group than those in the control group and Egr3^-/- group（P<0.05）, and were higher in the model group than those in the Egr3^-/-+model group（P<0.05）. The relative expression of Egr3 protein in aortic tissues increased gradually in the Egr3^-/- group, Egr3^-/-+model group, control group, and model group（P<0.05）. The relative expression of Bax protein in aortic tissues was higher in the model group and Egr3^-/-+model group than that in the control group and Egr3^-/- group（P<0.05）, and was higher in the model group than that in the Egr3^-/-+model group（P<0.05）. The relative expression of Bcl-2 protein was lower in the model group and Egr3^-/-+model group than that in the control group and Egr3^-/- group（P<0.05）, and was lower in the model group than that in the Egr3^-/-+model group（P<0.05）. Conclusion Egr3 is upregulated in aortic tissues of atherosclerosis mice, and to knockout the Egr3 gene can alleviate atherosclerotic lesions, which may be correlated with the inhibition of inflammation and apoptosis in aortic tissues.更多还原