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

作　　者：黄丽嫣 唐蕾 朱晓琳[2] 单云 史俊[3] 俞曼殊 孙谨怡 盛莉 盛梅笑[1] HUANG Liyan;TANG Lei;ZHU Xiaolin;SHAN Yun;SHI Jun;YU Manshu;SUN Jinyi;SHENG Li;SHENG Meixiao(Department of Nephrology,Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China;De-partment of Nephrology,the Second Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210017,China;School of Integrated Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 210023,China)

机构地区：[1]南京中医药大学附属医院肾科,江苏南京210029 [2]南京中医药大学第二附属医院肾科,江苏南京210017 [3]南京中医药大学中医学院·中西医结合学院,江苏南京210023

出　　处：《中国病理生理杂志》2023年第5期884-892,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82074351;No.82104754);江苏省中医院高峰学术人才项目(No.y2021rc10);江苏省研究生科研创新计划(No.KYCX22-1904;No.KYCX21-1660)。

摘　　要：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.

关 键 词：腹膜血管内皮细胞 血管新生 微血管周细胞 Ang1/Tie2信号通路 

分 类 号：R363.2[医药卫生—病理学] R365[医药卫生—基础医学]