NGF对脑梗死患者内皮祖细胞的影响及其机制研究
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摘要
第一章人外周血内皮祖细胞分离及鉴定
目的:
熟练掌握密度梯度离心法,能成功从人外周血中分离获得单个核细胞(MNCs)诱导分化为内皮祖细胞(endothelial progenitor cellsEPCs)。为一步研究提供可靠的细胞来源。
方法:
①使用EDTA无菌抗凝负压管从健康成人肘静脉抽血10ml,用密度梯度离心法获取单个核细胞;
②将分离的单个核细胞接种在人纤维连接蛋白包被的6孔细胞培养板,经诱导分化为内皮祖细胞,分别观察刚分离、培养4天、培养7天、培养14天的细胞形态;
③培养7天后,收集贴壁细胞,采用激光共聚焦显微镜(laserscanning confocal microscope,LSCM)鉴定FITC-UEA-I和Dil-acLDL双染阳性细胞为正在分化的EPCs;流式细胞仪检测细胞表型;
④将EPCs接种于将ECMatrixTM胶上,培养21天,观察生成血管情况。
结果:
①显微镜下细胞形态学特征:刚分离细胞呈圆形不贴壁;培养第4天细胞呈椭圆形、短梭形;培养第7天,出现多个细胞团,中间为圆形细胞,贴壁的梭型细胞从细胞团的周围向外生长;第14天左右可见首尾相连形成条索状结构;
②用Dil-acLDL和FITC-UEA-I对细胞染色后,通过激光共聚焦显微镜鉴定。Dil-acLDL和FITC-UEA-I双染色阳性细胞被认为是正在分化的EPCs;
③流式细胞仪检测细胞表面标志,结果显示贴壁细胞中表达CD34占(32.15±8.68)%,表达CD133占(18.73±7.12)%,表达KDR占(69.45±8.21)%,进一步证明了获得的细胞为EPCs;
④细胞在体外血管生成实验中可形成复杂的管腔样结构。
结论:
密度梯度离心法分离外周血单个核细胞,是有效获得内皮祖细胞的一条简单的途径。为内皮祖细胞实验研究提供了细胞基础。
第二章NGF对脑梗死患者外周血内皮祖细胞数量及部分生物学功能影响
目的
观察脑梗死患者外周血内皮祖细胞数量、生物学功能改变及神经生长因子(nerve growth factor,NGF)对其数量及生物学功能影响。
方法
(1)脑梗死患者外周血内皮祖细胞数量及生物学功能:
MTT法测定细胞增殖能力,采用改良的Boyden小室测定观察细胞的迁移和黏附能力,体外血管生成实验观察细胞在体外生成血管的能力。
(2)NGF对外周血内皮祖细胞数量及生物学功能影响:
不同浓度的NGF(20μg/L、40μg/L、60μg/L、80μg/L)培养外周血内皮祖细胞24h,观察外周血内皮祖细胞数量及迁移、黏附、增殖、体外血管形成的变化;60μg/L NGF培养外周血内皮祖细胞不同时间(0h,6h,12h,24h,48h),观察外周血内皮祖细胞数量及迁移、黏附、增殖、体外血管形成的变化。
(3)NGF对脑梗死患者外周血内皮祖细胞数量及生物学功能的影响:
NGF60μg/L培养脑梗死患者外周血内皮祖细胞24h,观察内皮祖细胞数量及迁移、黏附、增殖、体外血管形成的变化。
倒置显微镜对每孔的内皮祖细胞进行计数,改良的Boyden小室检测内皮祖细胞的迁移能力;黏附能力实验检测内皮祖细胞的粘附能力;体外血管生成试剂盒检测内皮祖细胞的血管生成能力;MTT比色法检测内皮祖细胞的增殖能力。
结果
①脑梗死患者外周血内皮祖细胞数量及生物学功能改变:脑梗死患者外周血内皮祖细胞数量较正常对照组显著减少,迁移、黏附、增殖、体外血管生成能力显著下降。
②NGF外周血内皮祖细胞数量及生物学功能影响:NGF显著增加内皮祖细胞数量及迁移、黏附、增殖、体外血管生成能力,在一定范围内呈一定的量效时效关系。NGF60μg/L培养外周血内皮祖细胞24h达到最大效应。
③NGF60μg/L培养外脑梗死患者周血内皮祖细胞24h,细胞数目及迁移、黏附、增殖、体外血管生成能力显著改善。
结论
脑梗死患者外周血内皮祖细胞数量较正常对照组显著减少,迁移、黏附、增殖、体外血管生成能力显著下降;NGF增加脑梗死患者外周血内皮祖细胞数量,改善细胞迁移、黏附、增殖、体外血管生成能力。
第三章NGF对脑梗死患者EPCs细胞PKB/eNOS信号通路影响
目的:
观察神经生长因子(NGF)对脑梗死患者外周血内皮祖细胞(endothelial progenitor cells)PKB/eNOS信号通路的影响,探讨NGF影响内皮祖细胞数量及生物学功能的分子机制。
方法
(1)脑梗死组、NGF、LY294002对EPCs细胞PKB总蛋白及磷酸化蛋白表达的影响。
(2)脑梗死组、NGF、LY294002及L-NAME对EPCs细胞eNOS总蛋白及磷酸化蛋白表达的影响。
(3)LY294002及L-NAME对脑梗死患者外周血EPCs数目、黏附、迁移、增殖能力及成血管数目的影响及细胞上清中NO的影响。
MTT法测定细胞增殖能力,采用改良的Boyden小室测定观察EPCs的迁移和黏附能力,体外血管生成实验观察EPCs在体外生成血管的能力。
(4)重氮还原法测定脑梗死组、脑梗死+NGF EPCs细胞上清中NO的影响。
结果
(1)脑梗死患者外周血EPCs细胞eNOS磷酸化蛋白显著降低,NGF可上调eNOS磷酸化,L-NAME可抑制NGF可上调PKB磷酸化的作用。
(2)脑梗死患者外周血EPCs细胞PKB磷酸化蛋白显著降低,NGF可上调PKB磷酸化,LY294002可抑制NGF可上调PKB磷酸化的作用。
(3)脑梗死患者外周血内皮祖细胞数量较正常对照组显著减少,迁移、黏附、增殖、体外血管生成能力显著下降。LY294002或L-NAME预处理后,脑梗死患者外周血内皮祖细胞数目明显减少,黏附、迁移、增殖能力及成血管能力降低更加显著,细胞NO的产生减少。
(4)脑梗死患者外周血内皮祖细胞细胞上清液中NO的浓度较对照组显著下降,LY294002及L-NAME加重NO浓度降低。
结论
脑梗死患者外周血EPCs细胞EPCs细胞PKB/eNOS信号通路活性下降,NO产生减少是内皮祖细胞数量及黏附、迁移、增殖能力及成血管能力降低的机制之一,NGF通过改善PKB/eNOS的磷酸化,上调内皮祖细胞数目及黏附、迁移、增殖能力及成血管功能。
Objective
To master expertly in isolation of mononuclear cells(MNCs) from Human peripheral blood by Ficoll density gradient centrifugation and to induct and different endothelial progenitor cells (EPCs) . Find a way to gain endothelial progenitor cells and to establish a consistence foundation to the next study.
Methods
(1) The sterility anticoagulation under-pressure tube with EDTA was used to draw blood 10ml from ulnar vein of healthy adult and MNCs were isolated from Human peripheral blood by Ficoll density gradient centrifugation.
(2) MNCs were cultured on the six well fibronectin-coated cell dishes and induced to EPCs. The change of the cellular shape was recorded after isolation at( 4days、7days、14days).
(3) EPCs were characterized as Dil-acLDL/FITC-UEA-1 double positive cell and detected by laser scanning Confocal microscope (LSCM).EPCs were further documented by demonstrating the expression of KDR,CD133 and CD34 with flow cytometry.
(4) EPCs were cultivated on the ECMatrixTM . After 21 days, the shape of the vessels was observed.
Results
(1) The character of EPCs appearance: cells that just isolating from blood can't adherence and the shape of those cell appears round. At 4days, the round cell transform into ellipse or short Fusiform. At 7days, lots of cell colony were observed , round cell at the middle of cell colony and Fusiform cell grow outside from center to surrounding. At 14days, cells connected with each other as trabs.
(2) The isolating cells were stained as Dil-acLDL/FITC-UEA-1 double positive cell detected by LSCM.
(3) EPCs were further documented by demonstrating the expression of KDR,CD133 and CD34 with flow cytometry. CD34 to occupy (32.15±8.68)%, CD133 occupy (18.73±7.12)%, KDR occupy (69.45±8.21)%. Further, the cells isolating from human peripheral blood were certified as EPCs.
(4) After 21 days, the shape of the vessels on the ECMatrixTM glue was observed.
Conclusions:
Ficoll density gradient centrifugation is a available method to isolate and identify endothelial progenitor cells from Human peripheral blood. It is a reliability way to gain endothelial progenitor cells and to establish a consistence foundation to the next study.
Objective
To investigate the number and adhesion,migration, in vitrovasculo genesis capacity of endothelial progenitor cells (EPCs) from cerebral infarction patients.Morever,the effect of nerve growth factor on the number and funcation of EPCs were evaluated.
Methods
(1) To evaluate the number and adhesion,migration, in vitrovasculo genesis capacity of endothelial progenitor cells (EPCs) from cerebral infarction patients
(2) the effect of nerve growth factor on EPCs from peripheral blood :EPCs were incubated with nerve growth factor in a series of concentrations (20ug/L、40ug/L、60ug/L、80ug/L) for 24 hours to choose the one affect the number and function of peripheral EPCs. EPCs were incubated with 60ug/L nerve growth factor for various time(0h,6h,12h,24h,48h) .
(3) the effect of nerve growth factor on EPCs from peripheral cerebral infarction patients:EPCs were incubated with nerve growth factor 60ug/L for 24 hours, then evaluate the number and function of EPCs.
EPCs proliferation were assayed with MTT. EPCs adhesion assay was performed by replating MNCs on culture dishes, and then the effect cells were counted. Migration were assayed with Boyden chamber. And in vitro vasculogenesis activity was assayed in vitro vasculogenesis kit.
Results
(1) The number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity were significantly impaired in cerebral infarction patients.
(2) Treating the EPCs with nerve growth factor, the number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity were significantly improved. 60ug/L nerve growth factor got to the peak after 24h (p < 0.01).
(3) Pretreated EPCs of cerebral infarction patients with 60ug/L nerve growth factor, the number and migration,adhesion,proliferation and in vitro vasculogenesis activity of EPCs were increased significantly.
Conclusion
Nerve growth factor increase the number of cultured EPCs and improve EPCs biological characteristics of cerebral infarction patients.
Objective
To investigate the effect of NGF on the expression AKT and eNOS, in cultured endothelial progenitor cells (EPCs) from peripheral blood of cerebral infarction patients, in order to study the mechanism of NGF on EPCs number and activity.
Methods
(1) EPCs of cerebral infarction patients were incubated with or without NGF LY294002 or L-NAME for 24 hours Expression eNOS, phosphorylation and eNOS were measured by Western Blot.
(2) EPCs of cerebral infarction patients were incubated with NGF LY294002 or L-NAME for 24 hours Expression PKB eNOS,phosphorylation of PKB and eNOS were measured by Western Blot.
(3) EPCs of cerebral infarction patients were incubated with NGF +LY294002 or L-NAME for 16 hours to To evaluate the number , function and NO of peripheral blood EPCs of cerebral infarction patients. EPCs proliferation were assayed with MTT. EPCs adhesion assay was performed by replating MNCs on culture dishes, and then the effect cells were counted. Migration were assayed with Boyden chamber. And in vitro vasculogenesis activity was assayed in vitro vasculogenesis kit. Concentration of NO were measured using Griess Reaction in cell culture supernatants.
(4) EPCs of cerebral infarction patients were incubated with or without NGF,Concentration of NO were measured using Griess Reaction in cell culture supernatants.
Results
(1) The expression of eNOS phosphorylation decreased in EPCs from peripheral cerebral infarction, NGF inproved eNOS phosphorylation without influence total eNOS. L-NAME inhibited phosphorylation of PKB, and also abolished the effect of NGF on L-NAME phos phorylation.
(2) The expression of PKB phosphorylation decreased in EPCs from peripheral cerebral infarction, NGF inproved PKB phosphorylation without influence total PKB. LY294002 inhibited phosphorylation of PKB, and also abolished the effect of NGF on PKB phosphorylation
(3) The number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity were significantly decreaseded in cerebral infarction patients. Treating the EPCs with either LY294002 or L-NAME , the number and functional activities of EPCs were significantly impaired comparable with these untreated. At same time the generation of NO decreased.
(4) Concentration of NO in EPCs culture supernatants decreased signicantly in cerebral infarction patients
Conclusion
Downregulation of PKB/eNOS pathway and decrease in NO generation was observed in EPCs in cerebral infarction patients,which appears to mediate the impairment number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity. NGF upreguation of PKB/eNOS phosphorylation and NO generation may potentially contributed to improvement of number and capacity of EPCs in in cerebral infarction patients.
目的:
熟练掌握密度梯度离心法,能成功从人外周血中分离获得单个核细胞(MNCs)诱导分化为内皮祖细胞(endothelial progenitor cellsEPCs)。为一步研究提供可靠的细胞来源。
方法:
①使用EDTA无菌抗凝负压管从健康成人肘静脉抽血10ml,用密度梯度离心法获取单个核细胞;
②将分离的单个核细胞接种在人纤维连接蛋白包被的6孔细胞培养板,经诱导分化为内皮祖细胞,分别观察刚分离、培养4天、培养7天、培养14天的细胞形态;
③培养7天后,收集贴壁细胞,采用激光共聚焦显微镜(laserscanning confocal microscope,LSCM)鉴定FITC-UEA-I和Dil-acLDL双染阳性细胞为正在分化的EPCs;流式细胞仪检测细胞表型;
④将EPCs接种于将ECMatrixTM胶上,培养21天,观察生成血管情况。
结果:
①显微镜下细胞形态学特征:刚分离细胞呈圆形不贴壁;培养第4天细胞呈椭圆形、短梭形;培养第7天,出现多个细胞团,中间为圆形细胞,贴壁的梭型细胞从细胞团的周围向外生长;第14天左右可见首尾相连形成条索状结构;
②用Dil-acLDL和FITC-UEA-I对细胞染色后,通过激光共聚焦显微镜鉴定。Dil-acLDL和FITC-UEA-I双染色阳性细胞被认为是正在分化的EPCs;
③流式细胞仪检测细胞表面标志,结果显示贴壁细胞中表达CD34占(32.15±8.68)%,表达CD133占(18.73±7.12)%,表达KDR占(69.45±8.21)%,进一步证明了获得的细胞为EPCs;
④细胞在体外血管生成实验中可形成复杂的管腔样结构。
结论:
密度梯度离心法分离外周血单个核细胞,是有效获得内皮祖细胞的一条简单的途径。为内皮祖细胞实验研究提供了细胞基础。
第二章NGF对脑梗死患者外周血内皮祖细胞数量及部分生物学功能影响
目的
观察脑梗死患者外周血内皮祖细胞数量、生物学功能改变及神经生长因子(nerve growth factor,NGF)对其数量及生物学功能影响。
方法
(1)脑梗死患者外周血内皮祖细胞数量及生物学功能:
MTT法测定细胞增殖能力,采用改良的Boyden小室测定观察细胞的迁移和黏附能力,体外血管生成实验观察细胞在体外生成血管的能力。
(2)NGF对外周血内皮祖细胞数量及生物学功能影响:
不同浓度的NGF(20μg/L、40μg/L、60μg/L、80μg/L)培养外周血内皮祖细胞24h,观察外周血内皮祖细胞数量及迁移、黏附、增殖、体外血管形成的变化;60μg/L NGF培养外周血内皮祖细胞不同时间(0h,6h,12h,24h,48h),观察外周血内皮祖细胞数量及迁移、黏附、增殖、体外血管形成的变化。
(3)NGF对脑梗死患者外周血内皮祖细胞数量及生物学功能的影响:
NGF60μg/L培养脑梗死患者外周血内皮祖细胞24h,观察内皮祖细胞数量及迁移、黏附、增殖、体外血管形成的变化。
倒置显微镜对每孔的内皮祖细胞进行计数,改良的Boyden小室检测内皮祖细胞的迁移能力;黏附能力实验检测内皮祖细胞的粘附能力;体外血管生成试剂盒检测内皮祖细胞的血管生成能力;MTT比色法检测内皮祖细胞的增殖能力。
结果
①脑梗死患者外周血内皮祖细胞数量及生物学功能改变:脑梗死患者外周血内皮祖细胞数量较正常对照组显著减少,迁移、黏附、增殖、体外血管生成能力显著下降。
②NGF外周血内皮祖细胞数量及生物学功能影响:NGF显著增加内皮祖细胞数量及迁移、黏附、增殖、体外血管生成能力,在一定范围内呈一定的量效时效关系。NGF60μg/L培养外周血内皮祖细胞24h达到最大效应。
③NGF60μg/L培养外脑梗死患者周血内皮祖细胞24h,细胞数目及迁移、黏附、增殖、体外血管生成能力显著改善。
结论
脑梗死患者外周血内皮祖细胞数量较正常对照组显著减少,迁移、黏附、增殖、体外血管生成能力显著下降;NGF增加脑梗死患者外周血内皮祖细胞数量,改善细胞迁移、黏附、增殖、体外血管生成能力。
第三章NGF对脑梗死患者EPCs细胞PKB/eNOS信号通路影响
目的:
观察神经生长因子(NGF)对脑梗死患者外周血内皮祖细胞(endothelial progenitor cells)PKB/eNOS信号通路的影响,探讨NGF影响内皮祖细胞数量及生物学功能的分子机制。
方法
(1)脑梗死组、NGF、LY294002对EPCs细胞PKB总蛋白及磷酸化蛋白表达的影响。
(2)脑梗死组、NGF、LY294002及L-NAME对EPCs细胞eNOS总蛋白及磷酸化蛋白表达的影响。
(3)LY294002及L-NAME对脑梗死患者外周血EPCs数目、黏附、迁移、增殖能力及成血管数目的影响及细胞上清中NO的影响。
MTT法测定细胞增殖能力,采用改良的Boyden小室测定观察EPCs的迁移和黏附能力,体外血管生成实验观察EPCs在体外生成血管的能力。
(4)重氮还原法测定脑梗死组、脑梗死+NGF EPCs细胞上清中NO的影响。
结果
(1)脑梗死患者外周血EPCs细胞eNOS磷酸化蛋白显著降低,NGF可上调eNOS磷酸化,L-NAME可抑制NGF可上调PKB磷酸化的作用。
(2)脑梗死患者外周血EPCs细胞PKB磷酸化蛋白显著降低,NGF可上调PKB磷酸化,LY294002可抑制NGF可上调PKB磷酸化的作用。
(3)脑梗死患者外周血内皮祖细胞数量较正常对照组显著减少,迁移、黏附、增殖、体外血管生成能力显著下降。LY294002或L-NAME预处理后,脑梗死患者外周血内皮祖细胞数目明显减少,黏附、迁移、增殖能力及成血管能力降低更加显著,细胞NO的产生减少。
(4)脑梗死患者外周血内皮祖细胞细胞上清液中NO的浓度较对照组显著下降,LY294002及L-NAME加重NO浓度降低。
结论
脑梗死患者外周血EPCs细胞EPCs细胞PKB/eNOS信号通路活性下降,NO产生减少是内皮祖细胞数量及黏附、迁移、增殖能力及成血管能力降低的机制之一,NGF通过改善PKB/eNOS的磷酸化,上调内皮祖细胞数目及黏附、迁移、增殖能力及成血管功能。
Objective
To master expertly in isolation of mononuclear cells(MNCs) from Human peripheral blood by Ficoll density gradient centrifugation and to induct and different endothelial progenitor cells (EPCs) . Find a way to gain endothelial progenitor cells and to establish a consistence foundation to the next study.
Methods
(1) The sterility anticoagulation under-pressure tube with EDTA was used to draw blood 10ml from ulnar vein of healthy adult and MNCs were isolated from Human peripheral blood by Ficoll density gradient centrifugation.
(2) MNCs were cultured on the six well fibronectin-coated cell dishes and induced to EPCs. The change of the cellular shape was recorded after isolation at( 4days、7days、14days).
(3) EPCs were characterized as Dil-acLDL/FITC-UEA-1 double positive cell and detected by laser scanning Confocal microscope (LSCM).EPCs were further documented by demonstrating the expression of KDR,CD133 and CD34 with flow cytometry.
(4) EPCs were cultivated on the ECMatrixTM . After 21 days, the shape of the vessels was observed.
Results
(1) The character of EPCs appearance: cells that just isolating from blood can't adherence and the shape of those cell appears round. At 4days, the round cell transform into ellipse or short Fusiform. At 7days, lots of cell colony were observed , round cell at the middle of cell colony and Fusiform cell grow outside from center to surrounding. At 14days, cells connected with each other as trabs.
(2) The isolating cells were stained as Dil-acLDL/FITC-UEA-1 double positive cell detected by LSCM.
(3) EPCs were further documented by demonstrating the expression of KDR,CD133 and CD34 with flow cytometry. CD34 to occupy (32.15±8.68)%, CD133 occupy (18.73±7.12)%, KDR occupy (69.45±8.21)%. Further, the cells isolating from human peripheral blood were certified as EPCs.
(4) After 21 days, the shape of the vessels on the ECMatrixTM glue was observed.
Conclusions:
Ficoll density gradient centrifugation is a available method to isolate and identify endothelial progenitor cells from Human peripheral blood. It is a reliability way to gain endothelial progenitor cells and to establish a consistence foundation to the next study.
Objective
To investigate the number and adhesion,migration, in vitrovasculo genesis capacity of endothelial progenitor cells (EPCs) from cerebral infarction patients.Morever,the effect of nerve growth factor on the number and funcation of EPCs were evaluated.
Methods
(1) To evaluate the number and adhesion,migration, in vitrovasculo genesis capacity of endothelial progenitor cells (EPCs) from cerebral infarction patients
(2) the effect of nerve growth factor on EPCs from peripheral blood :EPCs were incubated with nerve growth factor in a series of concentrations (20ug/L、40ug/L、60ug/L、80ug/L) for 24 hours to choose the one affect the number and function of peripheral EPCs. EPCs were incubated with 60ug/L nerve growth factor for various time(0h,6h,12h,24h,48h) .
(3) the effect of nerve growth factor on EPCs from peripheral cerebral infarction patients:EPCs were incubated with nerve growth factor 60ug/L for 24 hours, then evaluate the number and function of EPCs.
EPCs proliferation were assayed with MTT. EPCs adhesion assay was performed by replating MNCs on culture dishes, and then the effect cells were counted. Migration were assayed with Boyden chamber. And in vitro vasculogenesis activity was assayed in vitro vasculogenesis kit.
Results
(1) The number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity were significantly impaired in cerebral infarction patients.
(2) Treating the EPCs with nerve growth factor, the number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity were significantly improved. 60ug/L nerve growth factor got to the peak after 24h (p < 0.01).
(3) Pretreated EPCs of cerebral infarction patients with 60ug/L nerve growth factor, the number and migration,adhesion,proliferation and in vitro vasculogenesis activity of EPCs were increased significantly.
Conclusion
Nerve growth factor increase the number of cultured EPCs and improve EPCs biological characteristics of cerebral infarction patients.
Objective
To investigate the effect of NGF on the expression AKT and eNOS, in cultured endothelial progenitor cells (EPCs) from peripheral blood of cerebral infarction patients, in order to study the mechanism of NGF on EPCs number and activity.
Methods
(1) EPCs of cerebral infarction patients were incubated with or without NGF LY294002 or L-NAME for 24 hours Expression eNOS, phosphorylation and eNOS were measured by Western Blot.
(2) EPCs of cerebral infarction patients were incubated with NGF LY294002 or L-NAME for 24 hours Expression PKB eNOS,phosphorylation of PKB and eNOS were measured by Western Blot.
(3) EPCs of cerebral infarction patients were incubated with NGF +LY294002 or L-NAME for 16 hours to To evaluate the number , function and NO of peripheral blood EPCs of cerebral infarction patients. EPCs proliferation were assayed with MTT. EPCs adhesion assay was performed by replating MNCs on culture dishes, and then the effect cells were counted. Migration were assayed with Boyden chamber. And in vitro vasculogenesis activity was assayed in vitro vasculogenesis kit. Concentration of NO were measured using Griess Reaction in cell culture supernatants.
(4) EPCs of cerebral infarction patients were incubated with or without NGF,Concentration of NO were measured using Griess Reaction in cell culture supernatants.
Results
(1) The expression of eNOS phosphorylation decreased in EPCs from peripheral cerebral infarction, NGF inproved eNOS phosphorylation without influence total eNOS. L-NAME inhibited phosphorylation of PKB, and also abolished the effect of NGF on L-NAME phos phorylation.
(2) The expression of PKB phosphorylation decreased in EPCs from peripheral cerebral infarction, NGF inproved PKB phosphorylation without influence total PKB. LY294002 inhibited phosphorylation of PKB, and also abolished the effect of NGF on PKB phosphorylation
(3) The number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity were significantly decreaseded in cerebral infarction patients. Treating the EPCs with either LY294002 or L-NAME , the number and functional activities of EPCs were significantly impaired comparable with these untreated. At same time the generation of NO decreased.
(4) Concentration of NO in EPCs culture supernatants decreased signicantly in cerebral infarction patients
Conclusion
Downregulation of PKB/eNOS pathway and decrease in NO generation was observed in EPCs in cerebral infarction patients,which appears to mediate the impairment number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity. NGF upreguation of PKB/eNOS phosphorylation and NO generation may potentially contributed to improvement of number and capacity of EPCs in in cerebral infarction patients.
引文
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