高盐对血管内皮生长因子受体-3~+巨噬细胞表型及功能的影响
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摘要
目的观察高盐对血管内皮生长因子受体(VEGFR)-3~+巨噬细胞表型、淋巴管内皮细胞特性及功能的影响。方法利用流式分选将小鼠RAW264.7巨噬细胞中VEGFR-3~+亚群分选出来,分为Control组、低盐组(LS组,20mmol/L NaCl)和高盐组(HS组,40 mmol/L NaCl)。利用CCK-8法观察不同组VEGFR-3~+细胞活性;Real-time PCR检测NaCl干预后VEGFR-3~+巨噬细胞表型变化及淋巴管内皮细胞标志物mRNA表达水平;Transwell实验检测各组细胞的迁移功能,流式细胞术检测不同组细胞的吞噬能力。结果与Control组相比,高盐干预可以使VEGFR-3~+巨噬细胞的白细胞介素(IL)-1β、肿瘤坏死因子(TNF)-α、CC类趋化因子配体(CCL)2、血管内皮生长因子(VEGF)-C及张力应答性增强子结合蛋白(TonEBP)mRNA表达水平上调(P<0.05);HS组在高盐干预24、48 h后细胞活性均显著低于LS组(P<0.05);同时,HS组细胞迁移能力及细胞的吞噬能力与Control组相比显著增强,差异均有统计学意义(P<0.05)。结论高盐可使VEGFR-3~+巨噬细胞向M1型巨噬细胞偏移并表现出促淋巴管生成的特性,其迁移及吞噬能力显著增强,为进一步研究该亚群与淋巴管生成及心血管疾病的关系提供了依据。
Objective To investigate the effect of high salt concentration on the phenotypes, lymphatic endothelial cell characteristics and functions of endothelial growth factor receptor(VEGFR)-3~+macrophages.Methods The VEGFR-3~+subsets in mouse RAW264.7 macrophages were sorted by flow cytometry and divided into control group, low salt group(LS group, 20 mmol/L NaCl) and high salt group(HS group, 40 mmol/L NaCl). The viability of VEGFR-3~+cells was measured by CCK-8 method in different groups. Real-time PCR was used to detect the changes of VEGFR-3~+macrophage phenotypes and the mRNA expression levels of lymphatic endothelial cell markers after the intervention. Transwell assay was used to detect the migration of three groups. The phagocytosis ability was measured by flow cytometry.Results The levels of IL-1β, TNF-α, CCL2, VEGF-C and TonEBP mRNA were significantly increased in HS group compared with those of control group(P<0.05). After 24 h and 48 h high salt intervention, the viability of VEGFR-3~+macrophages was significantly decreased in HS group compared with those of LS group(P<0.05). Meanwhile, the migration and the phagocytosis ability were also significantly increased in HS group compared with those of control group(P<0.05).Conclusion The VEGFR-3~+macrophages can migrate to M1-type macrophages and show lymphangiogenesis-promoting characteristics after high salt intervention, which provides a theoretical basis for our further study about the relationships between this subpopulation and lymphangiogenesis and cardiovascular diseases.
Objective To investigate the effect of high salt concentration on the phenotypes, lymphatic endothelial cell characteristics and functions of endothelial growth factor receptor(VEGFR)-3~+macrophages.Methods The VEGFR-3~+subsets in mouse RAW264.7 macrophages were sorted by flow cytometry and divided into control group, low salt group(LS group, 20 mmol/L NaCl) and high salt group(HS group, 40 mmol/L NaCl). The viability of VEGFR-3~+cells was measured by CCK-8 method in different groups. Real-time PCR was used to detect the changes of VEGFR-3~+macrophage phenotypes and the mRNA expression levels of lymphatic endothelial cell markers after the intervention. Transwell assay was used to detect the migration of three groups. The phagocytosis ability was measured by flow cytometry.Results The levels of IL-1β, TNF-α, CCL2, VEGF-C and TonEBP mRNA were significantly increased in HS group compared with those of control group(P<0.05). After 24 h and 48 h high salt intervention, the viability of VEGFR-3~+macrophages was significantly decreased in HS group compared with those of LS group(P<0.05). Meanwhile, the migration and the phagocytosis ability were also significantly increased in HS group compared with those of control group(P<0.05).Conclusion The VEGFR-3~+macrophages can migrate to M1-type macrophages and show lymphangiogenesis-promoting characteristics after high salt intervention, which provides a theoretical basis for our further study about the relationships between this subpopulation and lymphangiogenesis and cardiovascular diseases.
引文
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[17]范志浩,李媛媛,李莉霞,等.维生素C对巨噬细胞增殖、迁移及吞噬功能的影响[J].中国临床解剖学杂志,2017,35(3):266-270.Fan ZH,Li YY,LI LX,et al.The effect of Vitamin C on macrophage proliferation,migration and phagocytic activity[J].Chinese Journal of Clinical Anatomy,2017,35(3):266-270.doi:10.13418/j.issn.1001-165x.2017.03.007.
[18]任海强,闫莉,李月,等.右美托咪定抑制炎症反应中巨噬细胞迁移[J].山西医科大学学报,2018,49(3):226-231.Ren HQ,Yan L,Li Y,et al.Inhibitory effect of dexmedetomidine on migration function of macrophage during the inflammatory reaction[J].Journal of Shanxi Medical University,2018,49(3):226-231.doi:10.13753/j.issn.1007-6611.2018.03.003.
[2]Yang GH,Zhou X,Ji WJ,et al.Overexpression of VEGF-Cattenuates chronic high salt intake-induced left ventricular maladaptive remodeling in spontaneously hypertensive rats[J].Am J Physiol Heart Circ Physiol,2014,306(4):H598-609.doi:10.1152/ajpheart.00585.2013.
[3]杨国红,李玉明.单核巨噬细胞特点及其盐敏感高血压靶器官损伤机制[J].武警医学,2017,28(12):1277-1281.Yang GH,Li YM.Characteristics of mononuclear macrophages and mechanism of salt-sensitive hypertensive target organ damage[J].Medical Journal of the Chinese People's Armed Police Force,2017,28(12):1277-1281.doi:10.14010/j.cnki.wjyx.2017.12.030.
[4]Murray PJ,Allen JE,Biswas SK,et al.Macrophage activation and polarization:nomenclature and experimental guidelines[J].Immunity,2014,41(1):14-20.doi:10.1016/j.immuni.2014.06.008.
[5]杨国红,周欣,刘军翔,等.高盐诱导的自发性高血压大鼠肾脏损害与外周血单核细胞亚群变化的相关性[J].中华高血压杂志,2018,26(3):225-232.Yang GH,Zhou X,Liu JX,et al.Correlation between peripheral blood monocyte subsets and renal injury in high-salt fed spontaneousl[J].Chinese Journal of Hypertension,2018,26(3):225-232.doi:10.16439/j.cnki.1673-7245.2018.03.009.
[6]杨国红,周欣,刘军翔,等.自发性高血压大鼠外周血单核细胞亚群与左室重塑的相关性研究[J].临床心血管病杂志,2018,34(5):513-518.Yang GH,Zhou X,Liu JX,et al.Correlation analysis between peripheral blood monocyte subsets and left ventricular remodeling in spontaneously hypertensive rats[J].Journal of Clinical Cardiology,2018,34(5):513-518.doi:10.13201/j.issn.1001-1439.2018.05.020.
[7]Heine GH,Ortiz A,Massy ZA,et al.Monocyte subpopulations and cardiovascular risk in chronic kidney disease[J].Nat Rev Nephrol,2012,8(6):362-369.doi:10.1038/nrneph.2012.41.
[8]Zhou X,Zhang L,Ji WJ,et al.Variation in dietary salt intake induces coordinated dynamics of monocyte subsets and monocyteplatelet aggregates in humans:implications in end organ inflammation[J].PLoS One,2013,8(4):e60332.doi:10.1371/journal.pone.0060332.
[9]梁艳红,张肇林,田铧,等.单核细胞向淋巴管内皮细胞诱导分化的潜能[J].中国组织工程研究与临床康复,2010,14(10):1897-1900.Liang YH,Zhang ZL,Tian H,et al.Differentiation potential of monocytes into lymphatic endothelial cells[J].Journal of Clinical Rehabilitative Tissue Engineering Research,2010,14(10):1897-1900.doi:10.3969/j.issn.1673-8225.2010.10.040.
[10]Makinen T,Veikkola T,Mustjoki S,et al.Isolated lymphatic endothelial cells transduce growth,survival and migratory signals via the VEGF-C/D receptor VEGFR-3[J].EMBO J,2001,20(17):4762-4773.doi:10.1093/emboj/20.17.4762.
[11]Machnik A,Neuhofer W,Jantsch J,et al.Macrophages regulate salt-dependent volume and blood pressure by a vascular endothelial growth factor-C-dependent buffering mechanism[J].Nat Med,2009,15(5):545-552.doi:10.1038/nm.1960.
[12]Titze J,Machnik A.Sodium sensing in the interstitium and relationship to hypertension[J].Curr Opin Nephrol Hypertens,2010,19(4):385-392.doi:10.1097/MNH.0b013e32833aeb3b.
[13]Machnik A,Dahlmann A,Kopp C,et al.Mononuclear phagocyte system depletion blocks interstitial tonicity-responsive enhancer binding protein/vascular endothelial growth factor C expression and induces salt-sensitive hypertension in rats[J].Hypertension,2010,55(3):755-761.doi:10.1161/HYPERTENSIONAHA.109.143339.
[14]Coffman TM.Under pressure:the search for the essential mechanisms of hypertension[J].Nat Med,2011,17(11):1402-1409.doi:10.1038/nm.2541.
[15]罗悦晨.电压门控型钠通道中介的单核/巨噬细胞免疫调节在心肌缺血再灌注损伤中的作用[D].天津:天津医科大学,2013.Luo YC.Role of Voltage-Gated sodium channels mediated immuno?modulation of monocytes/macrophages in myocardial ischemia re?perfusion injury[D].Tianjin:Tianjin Medical University,2013.
[16]Dmitrieva NI,Cai Q,Burg MB.Cells adapted to high Na Cl have many DNA breaks and impaired DNA repair both in cell culture and in vivo[J].Proc Natl Acad Sci USA,2004,101(8):2317-2322.doi:10.1037/pnas.0308463100.
[17]范志浩,李媛媛,李莉霞,等.维生素C对巨噬细胞增殖、迁移及吞噬功能的影响[J].中国临床解剖学杂志,2017,35(3):266-270.Fan ZH,Li YY,LI LX,et al.The effect of Vitamin C on macrophage proliferation,migration and phagocytic activity[J].Chinese Journal of Clinical Anatomy,2017,35(3):266-270.doi:10.13418/j.issn.1001-165x.2017.03.007.
[18]任海强,闫莉,李月,等.右美托咪定抑制炎症反应中巨噬细胞迁移[J].山西医科大学学报,2018,49(3):226-231.Ren HQ,Yan L,Li Y,et al.Inhibitory effect of dexmedetomidine on migration function of macrophage during the inflammatory reaction[J].Journal of Shanxi Medical University,2018,49(3):226-231.doi:10.13753/j.issn.1007-6611.2018.03.003.