A20基因转染对大鼠颈动脉再狭窄和核因子κB表达的影响

Effects of A20 Gene Transfection on Restenosis and Nuclear Factor-kappa B Expression of Rat Carotid Artery

目的观察局部转染锌指蛋白A20基因对大鼠颈动脉再狭窄模型球囊损伤后内膜增生和血管平滑肌细胞增殖的影响并探讨其可能的机制。方法建立大鼠颈总动脉球囊损伤模型。104只健康雄性SD大鼠随机分为4组(n=26):假手术组、模型组、对照组和治疗组。假手术组只进行颈动脉结扎,不进行球囊损伤术;模型组只进行球囊损伤术,不进行局部转染治疗;对照组球囊损伤术后局部灌注Lipofectamine 2000 Reagent+TE buffer;治疗组球囊损伤术后局部灌注Lipofectamine 2000 Reagent+pCAGGS-GFP/A20重组质粒。荧光显微镜观察A20在损伤动脉壁的转染效率;病理组织学观察内膜增生情况;溴脱氧尿嘧啶核苷标记技术评估血管平滑肌细胞的增殖指数;分别用免疫组织化学染色、Western-blotting方法检测核因子κB p65在各组大鼠颈总动脉中的表达情况。结果大鼠颈总动脉球囊损伤术后14 d模型组和对照组血管内膜显著增生。A20基因转染可抑制新内膜面积的增加(减少47.8%,P<0.05)和内/中膜比值的增加(减少42.9%,P<0.05)。术后10 d治疗组血管平滑肌细胞体内增殖指数(9.6%±2.3%)显著少于对照组(26.7%±5.1%,P<0.05)。术后10 d治疗组血管壁细胞核因子κB p65阳性细胞率显著低于对照组(P<0.05)。术后7 d、14 d和28 d治疗组血管组织核因子κB p65的蛋白表达量显著低于对照组(P<0.05)。结论局部转染A20基因可抑制损伤血管内膜增生及平滑肌细胞的增殖,其分子机制可能通过其负反馈抑制了核因子κB介导的胞内信号转导途径。

Aim To investigate the effects of in vivo local transfection of zinc finger protein A20 gene on restenosis of balloon injured rat carotid artery and its possible mechanism. Methods Balloon catheter denudation of the endothelium of rat common carotid artery was routinely used as a model of restenosis. 104 male Sprague-Dawley rats were randomly divided into 4 groups： the sham group（no injury）, the model group （the simple injury）, the control group （vacant transfection regent group） and the therapeutic group （A20 gene and transfection regent group）, pCAGGS-GFP/A20 （20 μg） with 40 μL Lipofectamine 2000 or TE buffered solution （20 μL） with 40 μL Lipofectamine 2000 was instilled into the lumen of the injured segment for 30 min after injury. The transfection efficiency of plasmid in injured vascular wall was evaluated 24 hours after transfection by using fluorescence microscope. Quantification of intimal hyperplasia was determined by pathologic examination. Proliferation index of VSMC in vivo was assessed by thymidine analogue bromodeoxyuridine （BrdU） labeling technique. The expression of nuclear factor-kappaB p65 （ NF-κBp65 ） of rat carotid arteries in different groups were confirmed by immunohistochemical staining and Western blot analysis. Results At day 14 significant intimal hyperplasia was detected after arterial injury in the model and control group. A20 gene transfection markedly reduced the neointimal area （47.8% reduction; P 〈 0.05） and intimal to media area ratio （42.9% reduction; P 〈 0.05） in the therapeutic group. Proliferation index of VSMC （BrdU index） at day 10 after operation was decreased significandy in the therapeutic group （9.6% ± 2.3% ） than in the control group （26.7% ± 5.1%, P 〈 0.05）. A signiflcandy lower level of NF-κBp65 positive ceils ratio was observed in die therapeutic group than in the control group at 10d after operation （ P 〈 0.05 ）. A significandy lower level of NF-κBp65 protein expression was observed in the therapeutic group than in the control group