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本研究旨在观察血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)及其受体阻断剂对SD大鼠胸主动脉外膜成纤维细胞亚群迁移及内皮素-1(endothelin-1,ET-1)表达的影响。采用克隆环法进行血管外膜成纤维细胞单克隆培养,并观察其细胞形态;RT-PCR方法进行细胞纯度鉴定;Transwell小室法检测AngⅡ对细胞亚群迁移的影响;荧光定量-PCR方法检测AngⅡ及其拮抗剂对preproET-1 mRNA表达的影响;ELISA法检测AngⅡ及其拮抗剂对ET-1分泌的影响。结果显示,克隆环法获得血管外膜成纤维细胞两个亚群:纺锤形细胞亚群和圆形细胞亚群。RT-PCR检测显示所获得的成纤维细胞亚群是纯细胞系。Transwell小室实验结果表明,两个细胞亚群均具备迁移能力,与自身对照相比,AngⅡ显著促进圆形细胞迁移,对纺锤形细胞迁移无明显影响;AngⅡ(1×10 8～1×10 6mol.L 1)对纺锤形细胞preproET-1 mRNA的表达及ET-1分泌无显著性影响,而呈浓度依赖性显著增加圆形细胞preproET-1mRNA的表达及ET-1的分泌(P<0.05,P<0.01);氯沙坦阻断AngⅡ诱导的圆形细胞preproET-1 mRNA的表达及ET-1的分泌,对纺锤形细胞preproET-1 mRNA的表达及ET-1的分泌无明显影响。以上结果提示,SD大鼠血管外膜成纤维细胞有两个细胞亚群:纺锤形细胞亚群和圆形细胞亚群。AngⅡ显著促进圆形细胞的迁移和ET-1的表达,提示两个细胞亚群的迁移机制可能不同,两种亚群在血管重建和修复过程中可能发挥不同作用。
The aim of this study was to investigate the effects of angiotensin Ⅱ (AngⅡ) and its receptor blockers on the migration of thoracic aorta fibroblasts and the expression of endothelin-1 (ET-1) The impact of expression. Monoclonal culture of vascular adventitial fibroblasts was performed by using the cloning loop method, and cell morphology was observed. Cell purity was identified by RT-PCR, Transwell chamber assay was used to detect the effect of AngⅡ on cell subpopulation migration, and fluorescence quantitative-PCR And its antagonist on preproET-1 mRNA expression; ELISA assay Ang Ⅱ and its antagonist on ET-1 secretion. The results showed that two subgroups of vascular adventitial fibroblasts were obtained by the cloning method: spindle-shaped cell subset and circular cell subset. RT-PCR assays showed that the fibroblast subpopulation obtained was a pure cell line. Transwell chamber results showed that both cell subpopulations had migratory ability. Compared with their own control, AngⅡ significantly promoted the migration of round cells and had no significant effect on the spindle cell migration. AngⅡ (1 × 10 8 ~ 1 × 10 6 mol .L 1) had no significant effect on the expression of preproET-1 mRNA and the secretion of ET-1 in spindle cells, but significantly increased the expression of preproET-1mRNA and the secretion of ET-1 in a dose-dependent manner (P <0.05, P <0.01). Losartan blocked the expression of preproET-1 mRNA and the secretion of ET-1 in round cells induced by AngⅡ, but had no effect on preproET-1 mRNA expression and ET-1 secretion in spindle cells. These results suggest that SD rat vascular adventitial fibroblasts have two cell subsets: spindle-shaped cell subpopulations and round cell subsets. AngⅡ significantly promoted the migration of round cells and the expression of ET-1, suggesting that the migration mechanisms of the two cell subpopulations may be different. The two subtypes may play different roles in the process of vascular reconstruction and repair.