血管紧张素Ⅱ及其受体拮抗剂对血管外膜成纤维细胞亚群增殖和胶原合成的影响

摘要

目的：探讨血管紧张素Ⅱ（ Ang Ⅱ）及其受体拮抗剂氯沙坦（ Los）和 PD123319对大鼠胸主动脉血管外膜成纤维细胞亚群增殖和胶原生成的影响。方法通过克隆环法获得血管外膜成纤维细胞单克隆，根据细胞表型克隆了两种细胞亚型并分别进行培养，逆转录聚合酶链反应（ RT-PCR）和免疫荧光染色方法鉴定细胞的纯度；流式细胞术对两细胞亚群的形状和大小进行比较；四甲基偶氮唑盐（ MTT）法和Western印迹法检测Ang Ⅱ及其受体拮抗剂Los、PD123319对细胞亚群的增殖活性和Ⅰ型胶原（ CollagenⅠ）合成的影响。结果克隆环法获得血管外膜成纤维细胞两个亚群，经显微镜和流式细胞仪观察根据细胞表型分别命名为：圆形细胞亚群（RC）和纺锤形细胞亚群（SC）。经 RT-PCR和免疫荧光染色对第Ⅷ因子（vWF）、CD8、MHC、Desmin进行检测，结果显示无内皮细胞、平滑肌细胞细胞标志物的表达，分别为两种纯系细胞。实验发现Ang Ⅱ对 RC亚群的增殖活性及CollagenⅠ的合成能力均有显著影响，既促进RC的增殖又提高I型胶原的合成，但经Los预处理后，AngⅡ的促增殖及胶原合成能力被显著性抑制（P＜0.05），而SC亚群的增殖活性和胶原合成情况无显著性改变。经PD123319预处理后，对RC亚群和SC亚群增殖和胶原的合成均无明显影响。结论 AngⅡ在促进两细胞亚群的增殖和胶原合成的能力方面有差异，说明血管外膜成纤维细胞两种亚群在表型、功能和作用机制上具有显著性的差异，提示两种细胞亚群在病理生理机制及其血管重构和修复过程中扮演着不同的角色。%Objective To explore the effect of angiotensin Ⅱand its receptor antagonist Losartan on cell proliferation and collagen synthesis in adventitial fibroblast subpopulations from rat thoracic aorta vascular.Methods Vascular adventitial fibroblasts were individually expanded by using cloning rings ,characterization of isolated cells were determined by reverse transcription-polymerase chain reaction ( RT-PCR) method and immunofluorescence staining.The size of subpopulations were compared by flow cytometry ,cells were treated with AngⅡor AngⅡplus Losartan ,PD123319 to detect cellular proliferation and collagen synthesis by MTT and Western blot.Results RT-PCR showed that fibroblast subpopulations did not have endothelial cells ,smooth muscle cells and leukocytes ,which nameed pure cell lines.Ang Ⅱ had different promotion on cell proliferation and collagen synthesis between two subpopulations ,but this effect could be significantly blocked with Losartan in round cells ,and had no effect in spindle cells.AngⅡalso had no significant effect on cell proliferation and collagen synthesis of two subpopulations with PD 123319.Conclusions AngⅡhas significant difference in adventitial fibroblast subpopulations from rat thoracic aorta vascular.It could promote cell proliferation and collagen synthesis of round cells ,and the effect is achieved by angiotensin Ⅱreceptor 1 (AT1),Ang Ⅱhas no significant effect on spindle cells.The conclusion showes that the two subpopulations of adventitial fibroblast have sig -nificant differences in phenotype ,function and mechanism of action ,which suggestes that the two cell subpopulations play different roles in the pathophysiological mechanisms of vascular remodeling and repair processes.