NR1D1在血管外膜成纤维细胞增殖和迁移中的作用

Role and mechanism of NR1D1 in proliferation and migration of vascular adventitial fibroblasts

目的探讨核受体亚家族1组D成员1(nuclear receptor subfamily 1,group D,member 1,NR1D1)在小鼠血管外膜成纤维细胞(adventitial fibroblasts,AFs)增殖和迁移中的作用及机制。方法培养C57BL/6J小鼠原代AFs,用携带Nr1d1基因的腺病毒转染AFs过表达NR1D1,用SKL2001恢复β-连环蛋白(β-catenin)表达。增殖细胞核抗原(Ki-67)细胞免疫荧光染色和CCK-8试剂盒用于检测细胞增殖,划痕实验用于检测细胞迁移速度。qPCR检测Nr1d1的mRNA水平,Western blot检测NR1D1、β-catenin蛋白水平。为了探究NR1D1在血管内膜增生中的作用,将20只雄性野生型C57BL/6J小鼠随机分为假手术组、颈动脉内皮损伤组、假手术+SR9009(NR1D1激动剂)组以及颈动脉内皮损伤+SR9009组,每组5只。造模完成后按组分别腹腔注射DMSO或SR9009(100 mg·kg^(-1)·d^(-1)),持续14 d。造模成功28 d后取材,HE染色观察颈动脉内膜增生程度。结果过表达NR1D1明显降低Ki-67阳性细胞率(P<0.01)及总细胞数目(P<0.01),并且使AFs划痕愈合速度减慢(P<0.01)。过表达NR1D1明显抑制β-catenin表达(P<0.05)。SKL2001恢复β-catenin表达后,过表达NR1D1对AFs增殖和迁移的抑制作用消失(P<0.01)。上调NR1D1活性能减轻颈动脉内皮损伤后内膜增生(P<0.01)。结论NR1D1可能通过抑制β-catenin表达,从而抑制小鼠AFs的增殖和迁移。

Aim To explore the role and mechanism of nuclear receptor subfamily 1,group D,member 1(NR1D1)in the proliferation and migration of mouse adventitial fibroblasts(AFs).Methods Primary AFs isolated from C57BL/6J mice were cultured.Adenovirus carrying Nr1d1 gene was used to overexpress NR1D1 in AFs.The expression ofβ-catenin was restored by SKL2001.Proliferating cell nuclear antigen(Ki-67)immunofluorescence staining and CCK-8 staining were used to determine cell proliferation,and scratch test was used to determine cell migration.qPCR was used to determine the mRNA level of Nr1d1.Western blot was used to determine the protein levels of NR1D1 andβ-catenin.To investigate the role of NR1D1 in intimal hyperplasia,20 male wild type C57BL/6J mice were randomly divided into sham group,carotid artery endothelial injury,sham+SR9009(NR1D1 agonist)group and carotid artery endothelial injury+SR9009(n=5 in each group).They were treated with DMSO or SR9009(100 mg·kg^(-1)·d^(-1))via intraperitoneal injection for 14 days after operation,respectively.The degree of carotid intimal hyperplasia was measured by HE staining 28 days after operation.Results NR1D1 overexpression significantly reduced the percentage of Ki-67-positive cells(P<0.01),total cell number(P<0.01)and slowed down the rate of wound-healing(P<0.01).NR1D1 overexpression significantly inhibited the expression ofβ-catenin(P<0.05).After the expression ofβ-catenin was restored by SKL2001,the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs were abolished(P<0.01).Enhanced activity of NR1D1 significantly ameliorated intimal hyperplasia after carotid endothelial injury(P<0.01).Conclusion NR1D1 may inhibit the proliferation and migration of AFs via suppressing the expression ofβ-catenin.