血管紧张素Ⅱ对骨髓源内皮祖细胞增殖能力的影响及机制的探讨

Effect of angiotensin Ⅱ on the proliferation of bone marrow-derived endothelial progenitor cells and possible mechanism

目的研究血管紧张素Ⅱ(AngⅡ)对骨髓源内皮祖细胞(EPCs)增殖能力的影响,并探讨其可能机制。方法通过密度梯度离心法分离大鼠骨髓单个核细胞,进行诱导分化,培养7天获得EPCs。抗AC133与抗血管内皮生长因子受体对EPCs双标后进行激光共聚焦鉴定。收集贴壁细胞加入不同浓度AngⅡ(10-7mol/L、10-8mol/L、10-9mol/L)进行干预,MTT法观察EPCs增殖能力的变化,并利用RT-PCR法观察不同浓度AngⅡ干预及缬沙坦、蛋白激酶C(PKC)抑制剂预处理后EPCs的Flk-1 mRNA表达量的变化。结果在血管内皮生长因子(VEGF)存在的前提下,AngⅡ可以增强EPCs的增殖能力,并可以上调Flk-1 mRNA的表达。缬沙坦、抑制剂可以显著抑制AngⅡ的这种作用。结论AngⅡ可以通过1型受体、PKC通路上调EPCs的Flk-1,在VEGF的作用下促进其增殖,从而有助于血管新生。

Objective To study the effect of angiotensin Ⅱ （Ang Ⅱ ）on the proliferation of bone marrow-derived endothelial progenitor cells （EPCs）and its possible mechanism. Methods Mononuclear ceils （MNCs） were isolated from bone marrow of rats by density gradient centrifugation. Then the MNCs were induced to differentiate into EPCs after 7 days of culture. EPCs were characterized as adherent cells double labeled with AC133 and Flk-1 by fluorescent staining and identified under laser scanning confocal microscope. Attached cells were collected and divided into different groups. A series of concentrations of Ang Ⅱ （10^-7 mol/L, 10^-8 mol/L, 10^-9 mol/L）were added to each group for intervention. The change in EPCs proliferative activity after the intervention was evaluated by MTT assay. RT-PCR was used to determine the change of Flk-1 mRNA expression in EPCs after the intervention by Ang Ⅱ at different concentrations or pretreatment with valsartan or GFX, an inhibitor of protein kinase C, before Ang Ⅱ stimulation. Results Ang Ⅱ enhanced the proliferative ability of EPCs in the presence of VEGF, and increased Flk-1 mRNA expression. The effects of Ang Ⅱ was blocked by valsartan or GFX. Conclusion It is suggested that AngⅡ may up-regulate Flk-1 mRNA expression of EPCs through Ang Ⅱ type 1 receptor and PKC pathway, which results in the proliferation of EPCs and then benefits vasculogenesis.