去铁胺促进血管内皮祖细胞靶向归巢和血管化的实验研究
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摘要
目的:探讨低氧模拟剂去铁胺(Desferrioxamine,DFO)模拟组织缺氧环境,促进血管内皮祖细胞(endothelial progenitorcells,EPCs)靶向归巢及促进血管化的可行性,并研究相关的信号通路。方法:分离、培养荧光素酶(luciferase)转基因Lewis大鼠的EPCs,作为移植细胞备用。实验动物NOD/SCID裸鼠被随机分为五组:对照组、EPCs、EPCsDFO、EPCs-DFO-AMD(AMD3100,趋化因子受体CXCR4抑制剂)、EPCs-DFO-LY(LY294002,磷酸肌醇3激酶(PI3K)抑制剂)组,每组10只。通过结扎裸鼠左侧股动脉造成左下肢缺血,经患侧股动脉移植EPCs,不同组别分别腹腔注射DFO(100mg/kg/d)、DFO和AMD3100(10mg/kg/d)、DFO和LY294002(20mg/kg/d),连续14d。分别从组织学,分子生物学,细胞学3个方面评价实验结果。结果:DFO的使用增加了裸鼠缺血下肢的血流灌注,改善了缺血下肢的功能,其新生毛细血管数量明显高于其他组,并且通过WesternBlot检测观察到缺血组织中VEGF(血管内皮生长因子),p-eNOS(磷酸化的一氧化氮合酶)和p-AKT(磷酸化蛋白激酶B)的分泌显著上调。在生物发光成像的研究中,DFO显著增加了EPCs向缺血患肢的迁移。这些作用均被PI3K抑制剂LY294002所抑制。结论:DFO的使用上调了EPCs向缺血患肢的迁移,改善了缺血患肢的血流灌注以及下肢功能,上调促血管生成因子的分泌,促进缺血组织的血管化,这些作用通过PI3K/AKT信号转导通路。
Objective To investigate whether desferrioxamine(DFO) can improve the homing of endothelial progenitor cells(EPCs)and improve neovascularization in ischemic hindlimb of rats. The relevant mechanism was also explored. Methods EPCs of luciferase transgenic Lewis rats were isolated and cultured as transplantation cells. Experimental animal NOD/SCID nude mice were randomly divided into five groups of 10: control, EPCs, EPCs-DFO, EPCs-DFO-AMD(AMD3100, CXCR4 inhibitor), and EPCs-DFO-LY(LY294002, the PI3 K inhibitor) groups. Left hindlimb ischemia was induced by ligating the left femoral artery in nude mice. EPCs were transplanted via the left femoral artery. After cell transplantation, intraperitoneal injection of DFO(100 mg/kg/d), DFO and AMD3100(10 mg/kg/d), DFO and LY294002(20 mg/kg/d),were performed respectively to different groups for 14 days. The experimental results were evaluated from three aspects: histology, molecular biology and cytology. Results DFO treatment increased the perfusion of ischemic hindlimb and improved the function of ischemic hindlimb in nude mice. The number of new capillaries was significantly higher than that of other groups. Moreover, VEGF, p-eNOS and p-AKT were significantly increased through Western Blotdetection. In bioluminescence imaging, DFO significantly increased the migration of EPCs to ischemic limbs. All these effects were diminished by LY294002. Conclusion DFO treatment increased the migration of EPCs to ischemic limbs, improved blood perfusion and hindlimb function in ischemic limbs, up-regulated the secretion of pro-angiogenic factors and promoted neovascularization of ischemic tissue. These effects are mediated by the PI3 K/AKT signal transduction pathway.
Objective To investigate whether desferrioxamine(DFO) can improve the homing of endothelial progenitor cells(EPCs)and improve neovascularization in ischemic hindlimb of rats. The relevant mechanism was also explored. Methods EPCs of luciferase transgenic Lewis rats were isolated and cultured as transplantation cells. Experimental animal NOD/SCID nude mice were randomly divided into five groups of 10: control, EPCs, EPCs-DFO, EPCs-DFO-AMD(AMD3100, CXCR4 inhibitor), and EPCs-DFO-LY(LY294002, the PI3 K inhibitor) groups. Left hindlimb ischemia was induced by ligating the left femoral artery in nude mice. EPCs were transplanted via the left femoral artery. After cell transplantation, intraperitoneal injection of DFO(100 mg/kg/d), DFO and AMD3100(10 mg/kg/d), DFO and LY294002(20 mg/kg/d),were performed respectively to different groups for 14 days. The experimental results were evaluated from three aspects: histology, molecular biology and cytology. Results DFO treatment increased the perfusion of ischemic hindlimb and improved the function of ischemic hindlimb in nude mice. The number of new capillaries was significantly higher than that of other groups. Moreover, VEGF, p-eNOS and p-AKT were significantly increased through Western Blotdetection. In bioluminescence imaging, DFO significantly increased the migration of EPCs to ischemic limbs. All these effects were diminished by LY294002. Conclusion DFO treatment increased the migration of EPCs to ischemic limbs, improved blood perfusion and hindlimb function in ischemic limbs, up-regulated the secretion of pro-angiogenic factors and promoted neovascularization of ischemic tissue. These effects are mediated by the PI3 K/AKT signal transduction pathway.
引文
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[13]邓蓉蓉,谢伊旻,谢林.间充质干细胞归巢的研究与进展[J].中国组织工程研究,2016,20(19):2879-2888.
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[16]Chan DA,Sutphin PD,Denko NC,et al.Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1alpha[J].J Biol Chem,2002,277(42):40112-40117.
[17]Zheng H,Fu G,Dai T,et al.Migration of endothelial progenitor cells mediated by stromal cell-derived Factor-1 alpha/CXCR4 via PI3K/Akt/eNOS signal transduction pathway[J].J Cardiovasc Pharmacol,2007,50(3):274-280.
[18]Jiang BH,Zheng JZ,Aoki M,et al.Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells[J].Proc Natl Acad Sci U S A,2000,97(4):1749-1753.
[19]Dimmeler S,Fleming I,Fisslthaler B,et al.Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation[J].Nature,1999,399(6736):601-605.
[20]Capoccia BJ,Shepherd RM,Link DC.G-CSF and AMD3100 mobilize monocytes into the blood that stimulate angiogenesis in vivo through a paracrine mechanism[J].Blood,2006,108(7):2438-2445.
[21]Peyvandi AA,Abbaszadeh HA,Roozbahany NA,et al.Deferoxamine promotes mesenchymal stem cell homing in noise-induced injured cochlea through PI3K/AKT pathway[J].Cell Prolif,2018,51(2):e12434.
[2]Schachinger V,Erbs S,Elsasser A,et al.Intracoronary bone marrowderived progenitor cells in acute myocardial infarction[J].N Engl JMed,2006,355(12):1210-1221.
[3]Ceradini DJ,Kulkarni AR,Callaghan MJ,et al.Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1[J].Nat Med,2004,10(8):858-864.
[4]Bergeron M,Yu AY,Solway KE,et al.Induction of hypoxia-inducible factor-1(HIF-1)and its target genes following focal ischaemia in rat brain[J].Eur J Neurosci,1999,11(12):4159-4170.
[5]Gray MJ,Zhang J,Ellis LM,et al.HIF-1alpha,STAT3,CBP/p300 and Ref-1/APE are components of a transcriptional complex that regulates Src-dependent hypoxia-induced expression of VEGF in pancreatic and prostate carcinomas[J].Oncogene,2005,24(19):3110-3120.
[6]Ratajczak MZ,Zuba-Surma E,Kucia M.The pleiotropic effects of the SDF-1-CXCR4 axis in organogenesis,regeneration and tumorigenesis[J].Leukemia,2006,20(11):1915-1924.
[7]Hou Z,Nie C,Si Z,et al.Deferoxamine enhances neovascularization and accelerates wound healing in diabetic rats via the accumulation of hypoxiainducible factor-1α[J].Diabetes Res Clin Pract,2013,101(1):62-71.
[8]Ikeda Y,Tajima S,Yoshida S,et al.Deferoxamine promotes angiogenesis via the activation of vascular endothelial cell function[J].Atherosclerosis,2011,215(2):339-347.
[9]Chekanov VS,Zargarian M,Baibekov I,et al.Deferoxamine-fibrin accelerates angiogenesis in a rabbit model of peripheral ischemia[J].Vasc Med,2003,8(3):157-162.
[10]Niiyama H,Huang NF,Rollins MD,et al.Murine model of hindlimb ischemia[J].J Vis Exp,2009,23(23):e1035.
[11]Krenning G,van Luyn MJ,Harmsen MC.Endothelial progenitor cellbased neovascularization:implications for therapy[J].Trends Mol Med,2009,15(4):180-189.
[12]Botta R,Gao E,Stassi G,et al.Heart infarct in NOD-SCID mice:therapeutic vasculogenesis by transplantation of human CD34+cells and low dose CD34+KDR+cells[J].FASEB J,2004,18(12):1392-1394.
[13]邓蓉蓉,谢伊旻,谢林.间充质干细胞归巢的研究与进展[J].中国组织工程研究,2016,20(19):2879-2888.
[14]Yamaguchi J,Kusano KF,Masuo O,et al.Stromal cell-derived factor-1effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization[J].Circulation,2003,107(9):1322-1328.
[15]Chiang KH,Cheng WL,Shih CM,et al.Statins,HMG-CoA reductase inhibitors,improve neovascularization by increasing the expression density of cxcr4 in endothelial progenitor cells[J].PLoS One,2015,10(8):e0136405.
[16]Chan DA,Sutphin PD,Denko NC,et al.Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1alpha[J].J Biol Chem,2002,277(42):40112-40117.
[17]Zheng H,Fu G,Dai T,et al.Migration of endothelial progenitor cells mediated by stromal cell-derived Factor-1 alpha/CXCR4 via PI3K/Akt/eNOS signal transduction pathway[J].J Cardiovasc Pharmacol,2007,50(3):274-280.
[18]Jiang BH,Zheng JZ,Aoki M,et al.Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells[J].Proc Natl Acad Sci U S A,2000,97(4):1749-1753.
[19]Dimmeler S,Fleming I,Fisslthaler B,et al.Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation[J].Nature,1999,399(6736):601-605.
[20]Capoccia BJ,Shepherd RM,Link DC.G-CSF and AMD3100 mobilize monocytes into the blood that stimulate angiogenesis in vivo through a paracrine mechanism[J].Blood,2006,108(7):2438-2445.
[21]Peyvandi AA,Abbaszadeh HA,Roozbahany NA,et al.Deferoxamine promotes mesenchymal stem cell homing in noise-induced injured cochlea through PI3K/AKT pathway[J].Cell Prolif,2018,51(2):e12434.