基于Ang1/Tie2信号通路研究周细胞调控腹膜血管新生的作用机制

Study on mechanism of pericyte-regulated peritoneal angiogenesis via Ang1/Tie2 signaling pathway

目的:本研究旨在通过观察血管周细胞与血管内皮细胞间的相互作用,探讨周细胞调控腹膜血管新生的作用机制。方法:使用转化生长因子β1(TGF-β1)干预人微血管周细胞(HMPCs)24、48和96 h,Western blot检测HMPCs中血管生成素1(Ang1)的表达。收集TGF-β1干预HMPCs后的上清液作为条件培养基(T-HMPCs-CM),用T-HMPCs-CM处理人腹膜血管内皮细胞(HPVECs)构建间接共培养模式;将TGF-β1处理过的HMPCs与HPVECs共同接种于基质胶上,构建直接共培养模式;以未经TGF-β1处理的HMPCs作为阴性对照组,以外源性Ang1处理的HPVECs作为阳性对照组,以人血管生成素受体Tie2抑制剂处理的HPVECs作为抑制剂组。采用CCK-8法、细胞划痕实验、Transwell实验、成管实验和细胞膜荧光探针染色法观察HPVECs增殖、迁移、血管生成情况,以及HPVECs与HMPCs共定位情况;Western blot检测HPVECs中Tie2蛋白及其磷酸化水平。结果:TGF-β1处理96 h,HMPCs中Ang1蛋白表达水平显著升高(P<0.01)。与阴性对照组相比,T-HMPCs-CM能够抑制HPVECs迁移、血管新生,并增加Tie2磷酸化以稳定血管状态,其作用类似于外源性Ang1;而使用Tie2抑制剂阻断Ang1/Tie2信号通路后,上述作用一定程度上被逆转。结论:TGF-β1可刺激HMPCs分泌Ang1,进而激活HPVECs的Tie2信号通路,从而抑制HPVECs血管新生,发挥维持腹膜血管稳定的作用。

AIM:To examine the mechanism by which pericytes regulate peritoneal angiogenesis by observing the interaction between vascular pericytes and vascular endothelial cells.METHODS:Transforming growth factor-β1(TGF-β1)was used to treat human microvascular pericytes(HMPCs)for 24,48 and 96 h,and the expression levels of an⁃giopoietin 1(Ang1)in HMPCs was detected by Western blot.The supernatant of TGF-β1-treated HMPCs was collected as conditioned medium(T-HMPC-CM),and human peritoneal vascular endothelial cells(HPVECs)were treated with THMPC-CM to construct an indirect co-culture model.HMPCs were treated with TGF-β1 and seeded with HPVECs on matrigel to construct a direct co-culture model.The HMPCs without TGF-β1 treatment served as the negative control group,the Ang1-treated HPVECs served as the positive control group,and the Tie2 inhibitor-treated HPVECs served as the inhibitor group.The proliferation,migration and angiogenesis of HPVECs were detected by CCK-8 assay,wound healing assay,Transwell assay and Tube formation assay,and the co-localization of HPVECs and HMPCs was observed by cell membrane fluorescence probe staining.Total and phosphorylated Tie2 protein levels were detected by Western blot.RE⁃SULTS:The expression level of Ang1 in HMPCs was increased significantly after treated with TGF-β1 for 96 h.Com⁃pared with negative control group,T-HMPC-CM inhibited the migration and angiogenesis of HPVECs,and Tie2 was phos⁃phorylated to stabilize the vascular state,which was similar to the effect of exogenous Ang1.After blocking the Ang1/Tie2 signaling pathway with a Tie2 inhibitor,these phenomena were reversed to some extent.CONCLUSION:The secretion of Ang1 in HMPCs is stimulated by TGF-β1,which activates the Tie2 signaling pathway in HPVECs,and then inhibits an⁃giogenesis of HPVECs,thus maintaining peritoneal vascular stability.