调节性T细胞促进球囊损伤颈动脉内皮化的作用及机制

doi:10.16098/j.issn.0529-1356.2019.05.005

摘要/Abstract

摘要：

目的探讨调节性T细胞（Treg细胞）促进球囊损伤颈动脉内皮化的作用及机制。方法使用Treg细胞分选试剂盒分选大鼠脾脏Treg细胞；构建大鼠颈动脉损伤模型；模型制作成功后分为对照组（尾静脉注射相同体积的生理盐水）、血管内皮生长因子（VEGF）组（尾静脉注射VEGF， 20 μmol/kg）和Treg细胞组（尾静脉注射1×105个Treg细胞）。HE染色检测内皮化情况；ELISA和免疫组织化学法检测血清中白细胞介素（IL）-10、转化生长因子-β（TGF-β）、IL-1β和肿瘤坏死因子α（TNF-α）的含量；流式细胞术检测单核细胞、T细胞和内皮祖细胞（EPC）的比例。结果组织学染色结果显示，对照组未见内皮细胞层的形成，Treg细胞组可见较多内皮细胞覆盖在颈动脉内层；免疫组织化学结果显示，对照组与Treg细胞组IL-10、TGF-β、IL-1β和TNF-α蛋白表达差异均有统计学意义（t=8.252，P<0.01；t=3.254，P<0.05；t=6.237，P<0.01；t=7.529，P<0.01）。ELISA结果显示，对照组IL-10、TGF-β、IL-1β和TNF-α的含量分别为（17.38±2.595）μg/L、（4.750±1.549）μg/L、（11.65±1.908）μg/L和（1.163±0.3333）μg/L；Treg细胞组的含量分别为（58.43±6.060）μg/L、（14.17±2.250）μg/L、（1.550±0.3819）μg/L和（0.2100±0.06938）μg/L，差异有显著性（t=6.170，P<0.01；t=3.558，P<0.01；t=5.191，P<0.01；t=2.800，P<0.05）；流式细胞术的结果显示，对照组CD34+VEGFR-2+EPC比例为（0.2838±0.01975）%，Treg细胞组为（0.5667±0.05993）%，差异有显著性（t=4.483，P<0.01）；IL-10阻断组EPC比例为（0.4807±0.03067）%，相对于Treg细胞组，差异不具有统计学意义（t=1.278，P>0.05）；TGF-β阻断组EPC比例为(0.3082±0.02291)%，相对于Treg细胞组，差异有显著性（t=4.029，P<0.01）。结论 Treg细胞通过诱导EPC动员，促进球囊损伤颈动脉的快速内皮化。

关键词: 调节性T细胞, 内皮祖细胞, 心脑血管疾病, 内皮化, 免疫组织化学, 大鼠

Abstract:

Objective To investigate the role and mechanism of regulatory T cells (Treg cell) in promoting endothelialization of carotid artery after balloon injury. Methods Treg cells were isolated from rat spleen by Treg cell sorting kit and carotid artery injury model was established. The rats were divided into three groups: control group (the same volume of normal saline), vascular endothelial growth factor (VEGF) group (intravenous injection of VEGF，20 μmol/kg) and Treg cell group (1×105 Treg cells injected via tail vein). HE staining and immunohistochemistry were used to detect endothelialization. The levels of interleukin (IL)-10, transforming growth factor β(TGF-β), IL-1β and tumor necrosis factor α (TNF-α)in serum were detected by ELISA. Flow cytometry was used to detect the proportion of monocytes, T cells and endothelial progenitor cells (EPC). Results Histological staining showed that no endothelial cells were observed in the control group, and more endothelial cells were found in the carotid intima in the Treg cell group. The result of immunohistochemistry showed that the expression of IL-10,TGF-β, IL-1 β and TNF-α protein in the control group and the Treg cell group were significantly different (t=8.252, P<0.01; t=3.254, P<0.05; t=6.237, P<0.01; t=7.529, P<0.01). ELISA result showed that the contents of IL-10, TGF-β, IL-1β and TNF-α in the control group were (17.38±2.595) μg/L, (4.750±1.549) μg/L, (11.65±1.908) μg/L and (1.163±0.3333)μg/L, respectively, while those in the Treg cell group were (58.43±6.060) μg/L, (14.17±2.250) μg/L, (1.550±0.3819) μg/L and (0.2100±0.06938) μg/L, with significant differences (t=6.170, P<0.01; t=3.558, P<0.01; t=5.191, P<0.01; t=2.800, P<0.05). The result of flow cytometry showed that the ratio of CD34+VEGFR-2+EPC in control group was (0.2838±0.01975)%, and that in Treg cell group was (0.5667±0.05993)% (t=4.483, P<0.01).The proportion of EPC in IL-10 blocking group was 0.4807±0.03067, compared with the Treg cell group, there was no significant difference (t=1.278, P>0.05).The proportion of EPC in TGF-β blocking group was(0.3082±0.02291)%, compared with the Treg cell group, the difference was significant (t=4.029, P<0.01). Conclusion Treg cells promote the rapid endothelialization of carotid artery after balloon injury by inducing EPC mobilization.

Key words:

Regulatory T cells, Endothelial progenitor cells, Cardiovascular and cerebrovascular diseases, Endothelialization, Immunohistochemistry, Rat

陈磊杨勇孟祥云秦立军. 调节性T细胞促进球囊损伤颈动脉内皮化的作用及机制[J]. 解剖学报, 2019, 50(5): 570-575.

CHEN Lei YANG Yong MENG Xiang-yun QIN Li-jun. Role and mechanism of regulatory T cells in promoting endothelialization of carotid artery after balloon injury[J]. Acta Anatomica Sinica, 2019, 50(5): 570-575.

参考文献

［1］ Zheng XT, Zeng RC, Huang JY, et al. The use of the angiosome concept for treating infrapopliteal critical limb ischemia through interventional therapy and determining the clinical significance of collateral vessels［J］. Ann Vasc Surg, 2016, 32(1):41-49.

［2］ Koyama A, Komori K, Otsuka R, et al. Dipeptidyl peptidase 4 inhibitor reduces intimal hyperplasia in rabbit autologous jugular vein graft under poor distal runoff［J］. J Vasc Surg, 2016, 63(5):1360-1370.

［3］ Qi W, Li Q, Liew CW, et al. SHP1 activation inhibits vascular smooth muscle cell proliferation and intimal hyperplasia in a rodent model of insulin resistance and diabetes.［J］. Diabetologia, 2017, 60(3):585-596.

［4］ Goldstone RN, Mccormack MC, Goldstein RL, et al. Photochemical tissue passivation attenuates AV fistula intimal hyperplasia［J］. Ann Surg, 2016, 267(1):183-188.

［5］ Boudaly S, Morin J, Berthier R, et al. Altered dendritic cells (DC) might be responsible for regulatory T cell imbalance and autoimmunity in nonobese diabetic (NOD) mice［J］. Eur Cytokine Netw, 2016, 13(1):29-37.

［6］ Li MO, Rudensky AY. T cell receptor signalling in the control of regulatory T cell differentiation and function.［J］. Nat Rev Immunol, 2016, 16(4):220-233.

［7］ Figueiredo AS, Schumacher A. The T helper type 17/regulatory T cell paradigm in pregnancy［J］. Immunology, 2016, 148(1):13-21.

［8］ Yang BH, Hagemann S, Mamareli P, et al. Foxp3(+) T cells expressing RORγt represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation.［J］. Mucosal Immunol, 2016, 9(2):444.

［9］ Malchow S, Leventhal DS, Lee Ⅴ, et al. Aire enforces immune tolerance by directing autoreactive T cells into the regulatory T cell lineage［J］. Immunity, 2016, 44(5):1102-1113. ［10］Tao JH, Cheng M, Tang JP, et al. Foxp3, regulatory T cell, and autoimmune diseases［J］. Inflammation, 2017, 40(1):328-339.

［11］Jeong-Su D, Anabelle Ⅴ, Osee SY, et al. An IL-27/Lag3 axis enhances Foxp3+regulatory T cell suppressive function and therapeutic efficacy［J］. Mucosal Immunol, 2016, 9(1):137-145.

［12］Esterházy D, Loschko J, London M, et al. Classical dendritic cells are required for dietary antigen-mediated peripheral regulatory T cell and tolerance induction［J］. Nat Immunol, 2016, 17(5):545-555.

［13］Kalekar LA, Schmiel SE, Nandiwada SL, et al. CD4+ T cell anergy prevents autoimmunity and generates regulatory T cell precursors［J］. Nat Immunol, 2016, 17(3):304-314.

［14］Huang ShL, Yang LX, Yang ShJ, et al. Role of ORAI1 in the balloon injury-induced rat carotid artery neointimal formation and the relationship between ORAI1 and sodium and calcium exchange 1［J］. Chinese J Cardiology, 2016, 44(11):968-972. (in Chinese)

黄世亮, 杨丽霞, 杨淑娟,等. ORAI1在损伤导致大鼠颈动脉新生内膜形成中的作用及其与钠钙交换体1的关系［J］. 中华心血管病杂志, 2016, 44(11):968-972.

［15］Erne P, Sudano Ⅰ, Resink TJ, et al. Interventional therapy for hypertension: back on track again ［J］ ? Crit Rev Clin Lab Sci, 2016, 54(1):18-25.

［16］Zheng XT, Zeng RC, Huang JY, et al. The use of the angiosome concept for treating infrapopliteal critical limb ischemia through interventional therapy and determining the clinical significance of collateral vessels ［J］. Ann Vasc Surg, 2016, 32(1):41-49.

［17］He M, Wang C, Sun JH, et al. Roscovitine attenuates intimal hyperplasia via inhibiting NF-κB and STAT3 activation induced by TNF-α in vascular smooth muscle cells［J］. Biochem Pharmacol, 2017, 137：51-60.

［18］Sun HJ,Zhao MX, Ren XS, et al. Salusin-β promotes vascular smooth muscle cell migration and Intimal hyperplasia after vascular injury via ROS/NFκB/MMP-9 pathway.［J］. Antioxid Redox Signal, 2016, 24(18):1045-1057.

［19］Marone G, Varricchi G, Loffredo S, et al. Mast cells and basophils in inflammatory and tumor angiogenesis and lymphangiogenesis［J］. Eur J Pharmacol, 2016, 778:146-151.

［20］Corliss BA, Azimi MS, Munson JM, et al. Macrophages: an inflammatory link between angiogenesis and lymphangiogenesis［J］. Microcirculation, 2016, 23(2):95-121.

［21］Zhang Z, Coutinho AE, Man TY, et al. Macrophage 11β-HSD-1 deficiency promotes inflammatory angiogenesis［J］. J Endocrinol, 2017, 234(3):291-299.

［22］Fortunel N, Hatzfeld JS, Monier M, et al. Release from quiescence of primitive human hematopoietic stem/progenitor cells by blocking their cell-surface TGF-β type Ⅱ receptor in a short-term in vitro assay［J］. Stem Cells, 2010, 18(2):102-111.

［23］Desouza CV, Hamel FG, Keshore B, et al. Role of inflammation and insulin resistance in endothelial progenitor cell dysfunction ［J］. Diabetes, 2011, 60(4):1286-1294.

［24］Ozkok A, Aktas E, Yilmaz A, et al. Decrease in endothelial progenitor cells associated with inflammation, but not with endothelial dysfunction in chronic hemodialysis patients［J］. Clin Nephrol, 2013, 79(1):21-30.

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