HGF促动脉血管形成的机理研究
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摘要
目的:
(1)确定Ad-HGF感染人股动脉内皮细胞(HFAECs)的最佳感染强度,初步探讨肝细胞生长因子(HGF)对HFAECs的Notch1和Dll4基因转录的调节作用,为进一步探讨HGF与Notch信号通路的关系奠定基础;
(2)探讨HGF对HFAECs的Dll4-Notch-Hey2信号通路的激活作用,及此通路激活后对HFAECs增殖和迁移能力的影响,从血管新生的角度来阐明Notch信号调控HGF促动脉形成的作用及机制。
(3)体外分离培养及鉴定人内皮祖细胞(EPCs),为下一步从血管发生的角度探讨HGF是否通过Notch信号通路调控祖细胞的分化来促动脉形成的机理提供体外细胞模型。
方法:
第一部分体外培养HFAECs,并用不同感染强度(MOI)(50,100,150,200,250,300pfu/cell)的携带绿色荧光蛋白基因的重组腺病毒(Ad-GFP)转染HFAECs,通过流式细胞仪(检测转染效率)和MTT法(检测细胞损伤程度)筛选和确定最佳MOI(高转染效率且对细胞低损伤),作为下一步携带HGF基因的重组腺病毒(Ad-HGF)转染细胞的最优条件。Ad-HGF以最佳的MOI转染HFAECs48h后,收集细胞上清,用ELISA法检测HGF蛋白的表达水平,选取HGF表达水平最佳的细胞提取总RNA,通过反转录PCR(RT-PCR)来检测Notch1和Dll4基因的转录水平。以转染Ad-GFP的细胞作为对照。
第二部分基于前期研究,Ad-HGF以最佳MOI(200 pfu/cell)转染HFAECs后0h、24h、48h、72h,分别收集细胞上清,用ELISA法检测不同时间点HGF蛋白的表达水平;同时收集细胞提取总RNA,通过RT-PCR检测不同时间点HFAECs中Notch1、Dll4和Hey2基因的转录水平;并采用MTT法和Transwell迁移实验分别观察HFAECs增殖和迁移能力的变化及与Dll4-Notch-Hey2信号通路激活的关系;均以转染Ad-GFP的细胞作对照。
第三部分无菌、肝素抗凝,取脐血(兰州军区总医院妇产科提供,产妇均知情同意)作为EPCs的标本来源。用PBS等倍稀释后取7 mL脐血缓慢加入装有3mL淋巴细胞分离液的离心管中,2000r/min离心25min,取云雾状灰白色层单个核细胞。将细胞接种在人纤维粘连蛋白包被的6孔板中,培养于M-199培养基中。观察贴壁细胞的形态变化;采用免疫细胞化学法鉴定EPCs。
实验数据采用SPSS13.0软件进行统计分析。
结果:
(1)以高转染效率且对细胞低损伤为标准,确定最佳MOI为200 pfu/cell;ELISA法检测HGF表达水平的结果显示,Ad-GFP组:(3.672±0.810) ng/ml;Ad-HGF组:(86.318±3.864)ng/ml。两组比较转染后48 h的HGF表达水平有统计学差异(P＜0.05);
(2) Ad-HGF转染HFAECs48 h后,RT-PCR结果显示,通过紫外凝胶分析仪,可观察到Ad-HGF组的Notch1和Dll4基因转录水平明显上调;应用凝胶成像分析系统分析凝胶成像图中目的基因的相对强度,结果显示:Ad-GFP组和Ad-HGF组的数据分别为:Dll4,0.788±0.021和0.936±0.018;Notch1,0和0.458±0.023,两组间进行单侧t检验分析,P＜0.05,有统计学意义。Ad-HGF组目的基因的相对强度明显高于Ad-GFP组;
(3) Ad-HGF转染HFAECs不同时间段后,ELISA检测HGF表达水平的结果显示:Ad-HGF有效的导入了细胞,且HGF表达水平48 h内有时间依赖性,72h开始下降;
(4) Ad-HGF转染HFAECs不同时间段后,RT-PCR的结果显示:通过紫外凝胶分析仪,发现在0h仅能检测到Dll4基因的转录,在48 h内Notch1和Dll4基因转录有时间依赖性,72h时Notch1基因转录已检测不到,Dll4基因转录也明显减弱。Hey2的转录水平的变化与Notch1相一致。凝胶成像分析系统软件处理结果与上述观察结果一致;
(5)在转染Ad-HGF不同时间段的HFAECs的MTT试验中初步观察到,与对照组相比,在各个时间点Ad-HGF组的HFAECs的增殖和存活能力明显增强;Transwell迁移实验结果表明,迁移介质中不含HGF时,两组细胞的迁移能力在各个时间点差异不明显(p＞0.05);迁移介质中含HGF时,在24h、48h Ad-HGF组细胞的迁移能力明显强于对照组(p＜0.05),且迁移能力在48h强于在24h但无统计学差异(p＞0.05);
(6) EPCs体外培养形态观察结果:细胞呈梭形,随培养时间的延长呈“铺路石”样,与有关文献报道相一致;
(7)免疫细胞化学法鉴定EPCs的结果显示:分离培养的细胞VEGFR-2和CD133均呈阳性反应,CD34反应较弱。
结论:
(1) HGF对HFAECs的Notch1受体和Dll4配体有诱导作用。暗示了HGF促动脉形成机制可能与Notch信号通路有一定的关联性;
(2) HGF可能通过激活动脉内皮细胞的Dll4-Notch-Hey2信号通路,间接地增强动脉内皮细胞增殖和迁移能力,来促进子代动脉分支的形成;
(3)采用密度梯度离心法从人脐血中分离培养的细胞具有EPCs特性,可作为我们所需的体外细胞模型。
Objective:
(1) To investigate the regulation of Dll4 and Notchl mediated by Hepatocyte growth factor(HGF) in human femoral artery endothelial cells(HFAECs),and to establish the basis for further investigating the mechanism of HGF promoting arteriogenesis;
(2) To investigate the relationship between the proliferation and migration ability of artery endothelial cells and Dll4-Notch-Hey2 signaling pathway in the process of arteriogenesis,and elucidate the mechanism of HGF promoting arteriogenesis.
(3) Endothelial progenitor cells(EPCs) were isolated,cultured and identified in vitro,and which were used as a study model from the point of angiogenesis,to further investigate that HGF promoted the formation of artery whether via Notch signal pathway regulating the differentiation of progenitor cells or not.
Methods:
Part 1 HFAECs were cultured in vitro and infected with the different multiplicity of infection(MOI) of Ad-GFP(50,100,150,200,250,300 pfu/cell).The optimal MOI was screened and determined by MTT(detecting the degree of cell damage) and flow cytometry(detecting the rate of transfection),and used as the optimal MOI of Ad-HGF.At 48 h after HFAECs were infected with the optimal MOI of Ad-HGF,HGF expression level in the supernatant was detected by ELISA;meanwhile,the transcription of Notchl and Dll4 genes was detected by reverse transcription-PCR(RT-PCR).Ad-GFP-infected HFAECs were used as negative controls.
Part 2 On account of prophase study,cells and supernatant were harvested at the indicated times(0,24,48,72 h) after HFAECs were infected with the 200 pfu/cell of Ad-HGF.HGF expression in the supernatant was detected by ELISA and the transcription of Notchl,Dll4 and Hey2 genes was analyzed by RT-PCR.The changes in the proliferation,survivorship and migration ability of HFAECs,and the relationship between Dll4-Notch-Hey2 signaling pathway and them was detected respectively by MTT and Transwell migration experiment.Ad-GFP-infected HFAECs were used as negative controls.
Part 3 Cord blood,asepstic,anticoagulated by Heparin,was used as the example resource of EPCs.After diluting with PBS(1:1),7 mL of cord blood was added slowly into centrifuge tube which containing 3mL of lymphocyte isolation.To centrifuge for 30 min at 2000 r/min and collect the mononuclearcells in cloudiness buffy coat.Cells were inoculated in 6-well plate coated with human plasma fibronectin purified protein and cultured in M-199 medium.To observe the morphologic change of adherent cells and identify EPCs by immunocytochemical method.
Experiment results were presented as mean±SD.Statistical analysis was performed using the SPSS 13.0 software package.
Results:
(1) The optimal MOI was 200 pfu/cell according to the high rate of transfection and the low degree of cell damage;the ELISA results showed that,Ad-GFP group: (3.672±0.810) ng/ml;Ad-HGF group:(86.318±3.864) ng/ml.At 48h after transfection,HGF expression level had statistical difference between two groups(P＜0.05);
(2) After Ad-HGF transfected HFAECs for 48h,the RT-PCR results showed that the transcription level of Notchl and Dll4 genes was obvious up-regulation in Ad-HGF group;the results of gel-image analytical system showed that Dll4: 0.788±0.021 and 0.936±0.018;Notchl:0 and 0.458±0.023 respectively in Ad-GFP and Ad-HGF groups,and the transcription level of objective genes had statistical significance(P＜0.05) by half t-test was performed between two groups.
(3) After Ad-HGF transfected HFAECs at the indicated times,the ELISA results showed that Ad-HGF effectually transduced into HFAECs,and HGF expression level had time-dependence within 48 h,and began to decrease at 72 h;
(4) After Ad-HGF transfected HFAECs at the indicated times,the RT-PCR results showed that the transcription of only Dll4 gene was detected at 0 h;the transcription of Notchl and Dll4 genes had time dependence within 48 h;the transcription of Notchl gene had not been detected and Dll4 weakened obviously at 72 h;kinetics of Hey2 induction by HGF was consistent with that of Notchl.The results of gel-image analytical system were consistent with above-mentioned observation results;
(5) In MTT experiment of Ad-HGF-infected HFAECs at the indicated times,it was initially observed comparing with the Ad-GFP group that the proliferation and survivorship ability of HFAECs enhanced obviously.The transwell migration experimental results showed that the migration ability of HFAECs between two groups was not significantly different in the absence of HGF at the indicated time (p＞0.05);but the migration ability of HFAECs in Ad-HGF group was obviously more enhanced than that in the control group in the presence of HGF at 24 h and 48 h(p＜0.05),and that at 48 h was more enhanced than at 24 h in Ad-HGF group but had no,significant difference(p＞0.05).
(6) The results of observing the morphous of EPCs cultured in vitro showed that cells were fusiform shape and the appearance of "stones paved roads" with the lasting of cultivation time,as was consistent with the report results of records.
(7) The result of identifying EPCs by immunocytochemical method showed that VEGFR-2、CD34 and CD133 of isolated cultured cells were all positive.
Conclusion:
(1)HGF had the up-regulation effect on the transcriptional level of Notchl and Dll4 genes in HFAECs:suggesting that the mechanism of HGF promoting arteriogenesis might be related with Notch signaling pathway.
(2) By activating Dll4-Notch-Hey2 signaling pathway,HGF indirectly prolonged cell survival,promoted the proliferation and migration ability of cells to promote the formation of offspring artery branching.
(3) Cells,which were isolated from cord blood by density gradient centrifugation and cultured,had the character of EPCs,and might be used as the further study model in vitro.
(1)确定Ad-HGF感染人股动脉内皮细胞(HFAECs)的最佳感染强度,初步探讨肝细胞生长因子(HGF)对HFAECs的Notch1和Dll4基因转录的调节作用,为进一步探讨HGF与Notch信号通路的关系奠定基础;
(2)探讨HGF对HFAECs的Dll4-Notch-Hey2信号通路的激活作用,及此通路激活后对HFAECs增殖和迁移能力的影响,从血管新生的角度来阐明Notch信号调控HGF促动脉形成的作用及机制。
(3)体外分离培养及鉴定人内皮祖细胞(EPCs),为下一步从血管发生的角度探讨HGF是否通过Notch信号通路调控祖细胞的分化来促动脉形成的机理提供体外细胞模型。
方法:
第一部分体外培养HFAECs,并用不同感染强度(MOI)(50,100,150,200,250,300pfu/cell)的携带绿色荧光蛋白基因的重组腺病毒(Ad-GFP)转染HFAECs,通过流式细胞仪(检测转染效率)和MTT法(检测细胞损伤程度)筛选和确定最佳MOI(高转染效率且对细胞低损伤),作为下一步携带HGF基因的重组腺病毒(Ad-HGF)转染细胞的最优条件。Ad-HGF以最佳的MOI转染HFAECs48h后,收集细胞上清,用ELISA法检测HGF蛋白的表达水平,选取HGF表达水平最佳的细胞提取总RNA,通过反转录PCR(RT-PCR)来检测Notch1和Dll4基因的转录水平。以转染Ad-GFP的细胞作为对照。
第二部分基于前期研究,Ad-HGF以最佳MOI(200 pfu/cell)转染HFAECs后0h、24h、48h、72h,分别收集细胞上清,用ELISA法检测不同时间点HGF蛋白的表达水平;同时收集细胞提取总RNA,通过RT-PCR检测不同时间点HFAECs中Notch1、Dll4和Hey2基因的转录水平;并采用MTT法和Transwell迁移实验分别观察HFAECs增殖和迁移能力的变化及与Dll4-Notch-Hey2信号通路激活的关系;均以转染Ad-GFP的细胞作对照。
第三部分无菌、肝素抗凝,取脐血(兰州军区总医院妇产科提供,产妇均知情同意)作为EPCs的标本来源。用PBS等倍稀释后取7 mL脐血缓慢加入装有3mL淋巴细胞分离液的离心管中,2000r/min离心25min,取云雾状灰白色层单个核细胞。将细胞接种在人纤维粘连蛋白包被的6孔板中,培养于M-199培养基中。观察贴壁细胞的形态变化;采用免疫细胞化学法鉴定EPCs。
实验数据采用SPSS13.0软件进行统计分析。
结果:
(1)以高转染效率且对细胞低损伤为标准,确定最佳MOI为200 pfu/cell;ELISA法检测HGF表达水平的结果显示,Ad-GFP组:(3.672±0.810) ng/ml;Ad-HGF组:(86.318±3.864)ng/ml。两组比较转染后48 h的HGF表达水平有统计学差异(P＜0.05);
(2) Ad-HGF转染HFAECs48 h后,RT-PCR结果显示,通过紫外凝胶分析仪,可观察到Ad-HGF组的Notch1和Dll4基因转录水平明显上调;应用凝胶成像分析系统分析凝胶成像图中目的基因的相对强度,结果显示:Ad-GFP组和Ad-HGF组的数据分别为:Dll4,0.788±0.021和0.936±0.018;Notch1,0和0.458±0.023,两组间进行单侧t检验分析,P＜0.05,有统计学意义。Ad-HGF组目的基因的相对强度明显高于Ad-GFP组;
(3) Ad-HGF转染HFAECs不同时间段后,ELISA检测HGF表达水平的结果显示:Ad-HGF有效的导入了细胞,且HGF表达水平48 h内有时间依赖性,72h开始下降;
(4) Ad-HGF转染HFAECs不同时间段后,RT-PCR的结果显示:通过紫外凝胶分析仪,发现在0h仅能检测到Dll4基因的转录,在48 h内Notch1和Dll4基因转录有时间依赖性,72h时Notch1基因转录已检测不到,Dll4基因转录也明显减弱。Hey2的转录水平的变化与Notch1相一致。凝胶成像分析系统软件处理结果与上述观察结果一致;
(5)在转染Ad-HGF不同时间段的HFAECs的MTT试验中初步观察到,与对照组相比,在各个时间点Ad-HGF组的HFAECs的增殖和存活能力明显增强;Transwell迁移实验结果表明,迁移介质中不含HGF时,两组细胞的迁移能力在各个时间点差异不明显(p＞0.05);迁移介质中含HGF时,在24h、48h Ad-HGF组细胞的迁移能力明显强于对照组(p＜0.05),且迁移能力在48h强于在24h但无统计学差异(p＞0.05);
(6) EPCs体外培养形态观察结果:细胞呈梭形,随培养时间的延长呈“铺路石”样,与有关文献报道相一致;
(7)免疫细胞化学法鉴定EPCs的结果显示:分离培养的细胞VEGFR-2和CD133均呈阳性反应,CD34反应较弱。
结论:
(1) HGF对HFAECs的Notch1受体和Dll4配体有诱导作用。暗示了HGF促动脉形成机制可能与Notch信号通路有一定的关联性;
(2) HGF可能通过激活动脉内皮细胞的Dll4-Notch-Hey2信号通路,间接地增强动脉内皮细胞增殖和迁移能力,来促进子代动脉分支的形成;
(3)采用密度梯度离心法从人脐血中分离培养的细胞具有EPCs特性,可作为我们所需的体外细胞模型。
Objective:
(1) To investigate the regulation of Dll4 and Notchl mediated by Hepatocyte growth factor(HGF) in human femoral artery endothelial cells(HFAECs),and to establish the basis for further investigating the mechanism of HGF promoting arteriogenesis;
(2) To investigate the relationship between the proliferation and migration ability of artery endothelial cells and Dll4-Notch-Hey2 signaling pathway in the process of arteriogenesis,and elucidate the mechanism of HGF promoting arteriogenesis.
(3) Endothelial progenitor cells(EPCs) were isolated,cultured and identified in vitro,and which were used as a study model from the point of angiogenesis,to further investigate that HGF promoted the formation of artery whether via Notch signal pathway regulating the differentiation of progenitor cells or not.
Methods:
Part 1 HFAECs were cultured in vitro and infected with the different multiplicity of infection(MOI) of Ad-GFP(50,100,150,200,250,300 pfu/cell).The optimal MOI was screened and determined by MTT(detecting the degree of cell damage) and flow cytometry(detecting the rate of transfection),and used as the optimal MOI of Ad-HGF.At 48 h after HFAECs were infected with the optimal MOI of Ad-HGF,HGF expression level in the supernatant was detected by ELISA;meanwhile,the transcription of Notchl and Dll4 genes was detected by reverse transcription-PCR(RT-PCR).Ad-GFP-infected HFAECs were used as negative controls.
Part 2 On account of prophase study,cells and supernatant were harvested at the indicated times(0,24,48,72 h) after HFAECs were infected with the 200 pfu/cell of Ad-HGF.HGF expression in the supernatant was detected by ELISA and the transcription of Notchl,Dll4 and Hey2 genes was analyzed by RT-PCR.The changes in the proliferation,survivorship and migration ability of HFAECs,and the relationship between Dll4-Notch-Hey2 signaling pathway and them was detected respectively by MTT and Transwell migration experiment.Ad-GFP-infected HFAECs were used as negative controls.
Part 3 Cord blood,asepstic,anticoagulated by Heparin,was used as the example resource of EPCs.After diluting with PBS(1:1),7 mL of cord blood was added slowly into centrifuge tube which containing 3mL of lymphocyte isolation.To centrifuge for 30 min at 2000 r/min and collect the mononuclearcells in cloudiness buffy coat.Cells were inoculated in 6-well plate coated with human plasma fibronectin purified protein and cultured in M-199 medium.To observe the morphologic change of adherent cells and identify EPCs by immunocytochemical method.
Experiment results were presented as mean±SD.Statistical analysis was performed using the SPSS 13.0 software package.
Results:
(1) The optimal MOI was 200 pfu/cell according to the high rate of transfection and the low degree of cell damage;the ELISA results showed that,Ad-GFP group: (3.672±0.810) ng/ml;Ad-HGF group:(86.318±3.864) ng/ml.At 48h after transfection,HGF expression level had statistical difference between two groups(P＜0.05);
(2) After Ad-HGF transfected HFAECs for 48h,the RT-PCR results showed that the transcription level of Notchl and Dll4 genes was obvious up-regulation in Ad-HGF group;the results of gel-image analytical system showed that Dll4: 0.788±0.021 and 0.936±0.018;Notchl:0 and 0.458±0.023 respectively in Ad-GFP and Ad-HGF groups,and the transcription level of objective genes had statistical significance(P＜0.05) by half t-test was performed between two groups.
(3) After Ad-HGF transfected HFAECs at the indicated times,the ELISA results showed that Ad-HGF effectually transduced into HFAECs,and HGF expression level had time-dependence within 48 h,and began to decrease at 72 h;
(4) After Ad-HGF transfected HFAECs at the indicated times,the RT-PCR results showed that the transcription of only Dll4 gene was detected at 0 h;the transcription of Notchl and Dll4 genes had time dependence within 48 h;the transcription of Notchl gene had not been detected and Dll4 weakened obviously at 72 h;kinetics of Hey2 induction by HGF was consistent with that of Notchl.The results of gel-image analytical system were consistent with above-mentioned observation results;
(5) In MTT experiment of Ad-HGF-infected HFAECs at the indicated times,it was initially observed comparing with the Ad-GFP group that the proliferation and survivorship ability of HFAECs enhanced obviously.The transwell migration experimental results showed that the migration ability of HFAECs between two groups was not significantly different in the absence of HGF at the indicated time (p＞0.05);but the migration ability of HFAECs in Ad-HGF group was obviously more enhanced than that in the control group in the presence of HGF at 24 h and 48 h(p＜0.05),and that at 48 h was more enhanced than at 24 h in Ad-HGF group but had no,significant difference(p＞0.05).
(6) The results of observing the morphous of EPCs cultured in vitro showed that cells were fusiform shape and the appearance of "stones paved roads" with the lasting of cultivation time,as was consistent with the report results of records.
(7) The result of identifying EPCs by immunocytochemical method showed that VEGFR-2、CD34 and CD133 of isolated cultured cells were all positive.
Conclusion:
(1)HGF had the up-regulation effect on the transcriptional level of Notchl and Dll4 genes in HFAECs:suggesting that the mechanism of HGF promoting arteriogenesis might be related with Notch signaling pathway.
(2) By activating Dll4-Notch-Hey2 signaling pathway,HGF indirectly prolonged cell survival,promoted the proliferation and migration ability of cells to promote the formation of offspring artery branching.
(3) Cells,which were isolated from cord blood by density gradient centrifugation and cultured,had the character of EPCs,and might be used as the further study model in vitro.
引文
[1] Ha XQ, et al.Therapeutic angiogenesis induced by human hepatocyte growth factor gene in dog hindlimb ischemia models. Chinese Science Bulletin , 2003,48(7):676-680.
[2] Wu DL, Ha XQ, et al.Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat myocardial ischemia models.Chinese Science Bulletin , 2003,48(3):273-276.
[3] Wu DL, Ha XQ, et al.Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat myocardial ischemia models.Chinese Science Bulletin, 2003,48(3):273-276.
[4] Ha XQ, et al.Gene therapy for pathological scar with hepatocyte growth factor mediated by recombinant adenovirus vector. Science in China, 2003,46(3):320-327.
[5] Serneels L, Dejaegere T, Craessaerts K, et al. Differential contribution of the three Aph1 genes to {gamma}-secretase activity in vivo. Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1719-24.
[6] Duarte A, Hirashima M, Benedito R, et al. Dosage-sensitive requirement for mouse D114 in artery development. Genes Dev. 2004 Oct 15;18(20):2474-8.
[7] Alva JA, Iruela-Arispe ML. Notch signaling in vascular morphogenesis. Curr Opin Hematol. 2004 Jul;11(4):278-83.
[8] Lawson ND, Scheer N, Pham VN, et,al. Notch signaling is required for arterial-venous differentiation during embryonic vascular development. Development. 2001 Oct;128(19):3675-83.
[9] Taylor KL, Henderson AM, Hughes CC. Notch activation during endothelial cell network formation in vitro targets the basic HLH transcription factor HESR-1 and downregulaties VEGFR-2/KDR expression. Microvasc Res. 2002,64(3):372-83.
[10] Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nature Med.2000, 6(3):389-395.
[11] Stella MC,Trusolino L,Pennacchietti S,et al. Negative feedback regulation of Met-dependent invasive growth by Notch. Mol Cell Biol.2005 May;25(10):3982-3996.
[12] Pires-daSilva A , Sommer RJ .The evolution of signalling pathways in animal development. Nat Rev Genet ,2003 ,4 (12) 39—49.
[13] Ha XQ, et al. Effect of human hepatocyte growth factor on promoting wound healing and preventing scar formation by adenovirus-mediated gene transfer. Chinese Medical Journal,2003,116(7): 1029-1033.
[14] Rossant J, Hirashima M. Vascular development and patterning: making the right choice. Curr Opin Genet Dev. 2003,13(4):408-12.
[15] Lawson ND, Vogel AM, Weinstein BM. sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev Cell. 2002 Jul;3(1): 127-36.
[16] Serneels L, Dejaegere T, Craessaerts K, et al. Differential contribution of the three Aph1 genes to {gamma}-secretase activity in vivo. Proc Natl Acad Sci U S A. 2005 Feb 1; 102(5): 1719-24.
[17] Duarte A, Hirashima M, Benedito R, et al. Dosage-sensitive requirement for mouse D114 in artery development. Genes Dev. 2004 Oct 15;18(20):2474-8.
[18] Alva JA, Iruela-Arispe ML. Notch signaling in vascular morphogenesis. Curr Opin Hematol. 2004 Jul;11(4):278-83.
[19] Shawber CJ, Kitajewski J. Notch function in the vasculature: insights from zebrafish, mouse and man. Bioessays. 2004 Mar;26(3):225-34.
[20]. T. Kadesch, Notch signaling: the demise of elegant simplicity.Curr Opin Genet Dev,2004, 14 (5): 506-512.
[21]. Luke T.Krebs,Yingzi Xue,Christine R.Norton,et al. Notch signaling is essential for vascular morphogenesis in mice .Genes Dev., 2000, 14(1): 1343-1352.
[22] C Mailhos, U Modlich, J Lewis, et al Delta4, an endothelial specific notch ligand expressed at sites of physiological and tumor angiogenesis.Differentiation, 2001, 69(2-3): 135-144.
[23] H Diez,A Fischer,A Winkler,et al . Hypoxia-mediated activation of D114-Notch-Hey2 signaling in endothelial progenitor cells and adoption of arterial cell fate. Exp Cell Res, 2007, 313(1): 1-9.
[24]. Liu ZJ, Shirakawa T, Li Y, et,al. Regulation of Notchl and D114 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis. Mol Cell Biol. 2003,23(1): 14-25.
[25].Scehnet JS,Jiang W,Kumar SR,et al.Inhibition of D114 mediated signaling induces proliferation of immature vessels and results in poor tissue perfusion. Blood, 2007,109:4753-4760.
[26] Hellstrom M,Phng LK,Hofmann JJ,et al.D114 signaling through Notch1 regulates formation of tip cells during angiogenesis. Nature, 2007,445(7129):776-780.
[31] Peichev M,Naiyer AJ,Pereira D, et al.Expression of VEGF-2 and AC133 by circulating human CD34~+ cells identifies a population of functional endothelial precursors[J]. Blood, 2000,95(3):952-958.
[32] Yin A,Miraglia S,Zarjani E,et al. AC133,a novel marker for human hematopoie stem and progenitor cells[J]. Blood, 1997,90:5002-5012.
[1]Iwaguro H,Asahara T.Endothelial progenitor cell culture and gene transfer[J].Methods Mol Med,2005,112:239-247.
[2]Ingram DA,Cap lice NM,Mervin C,et al.Unresolved questions,changing definitions,and novel paradigms for defining endothelial progenitor cells[J].Blood,2005,106(5):1525-1531.
[3]Fox A,Smythe J,Fisher N,et al.Mobilization of endothelial progenitor cells into the circulation[J].Anaesthesist,2007,5:42-47.
[4]Ho JW,Pang RW,Lau C,et al.Significance of circulating endothelial progenitor cells in hepatocellular carcinoma[J].Hepatology,2006,44(4):836-843.
[5]Christoph Kalka,Hassan Tehrani,Bemd Laudenberg,et al.VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease[J].Ann.Thorac.Surg.,2000;70(3):829-834.
[6]王改玲,肖传实,邱龄,等.粒细胞集落刺激因子对内皮前体细胞的动员[J].医学研究生学报,2006,19(4):3342-3371.
[7]肖刚峰,张怀勤,季亢挺,等.丹参素对体外培养内皮祖细胞数量和功能的影响[J].华西药学杂志,2006,21(3):2322-2341.
[8]Kerstin Strehlow,Nikos Werner,Jan Berweiler,et al.Estrogen Increases Bone Marrow-Derived Endothelial Progenitor Cell Production and Diminishes Neointima Formation[J].Circulation,2003;107(24):3059-3065.
[9]Askari AT,Unzek S,Popovic ZB,et al,Effect of stromal-cell-derived factor-1 on stem-cell homing and tissue regeneration in ischacmic cardiomyopathy[J].Lance,2003,362(9385):697-703.
[10]Murayama T,Tepper OM,Silver M,et al.Determination of bone marrow derived endothelial progenitor cell significance in angiogenic growth factor induced neovascularization in vivo[J].Exp Hematol,2002,30(8):967.
[11]Chavakis E,Aicher A,Heeschen C,et al.Role of β2-integrins for homing and neovascularization capacity of endothelial progenitor cells[J].Exp Med,2005,201(1):63-72.
[12]Verama S,Kuliszewski MA,Li SH,et al.C-reactive protein attenuates endothelial progenitor cell survival,differentiation,and function:further evidence of a mechanistic link between C-reactive protein and cardiovascular disease[J].Circulation,2004,109(17):2058.
[13]Urbich C,Heeschen C,Aicher A,et al.Cathepsin-L is required for endothelial progenitor cell induced neovascularization[J].Nat Med,2005,11(2):206-213.
[14]Hur J,Yoon CH,Lee CS,et al.Akt is a key modulator of endothelial progenitor cell trafficking in ischemic muscle[J].Stem Cells,2007,25(7):1769-1778.
[15]Dirk It.Walter,Kilian Rittig,Ferdinand H.Bahlmann,et al.Statin Therapy Accelerates Reendothelialization:A Novel Effect Involving Mobilization and Incorporation of Bone Marrow-Derived Endothelial Progenitor Cells [J]. Circulation,2002;105(25):3017-3024.
[16] Ying Guo, Rebecca Chan, Heather Ramsey,et al. The homeoprotein Hex is required for hemangioblast differentiation[J].Blood, 2003;102(7):2428-2435.
[17] Landmeser U , Eug berdiug N , Bahlmann FH , et al. Statin-induced improvement of endothelial progenitor cell mobilization , myocardial neovascularization , left ventricular function and survival after experimental myocardial infarction requires endothelial nitricoxide synthase[ J ].Circulation , 2004 , 110(14): 1933.
[18] Blann AD ,Pretorius A. Circulating endothelial cells and endothelial progenitor cells :Two sides of the same coin or two different coins [ J ] . Atherosclerosis , 2006 , 188 (1) :12-18.
[19] Schmidt-Lucke C, Rossig L, Fichtlscherer S, et al. Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair[ J ]. Circulation.2005;111(22):2981-2987.
[20] Gian Paolo Fadini, Saverio Sartore, Mattia Albiero, et al.Number and Function of Endothelial Progenitor Cells as a Marker of Severity for Diabetic Vasculopathy[ J ].Arterioscler. Thromb. Vasc. Biol., 2006; 26(9): 2140 - 2146.
[21] Hon-Kan Yip, Li-Teh Chang, Wen-Neng Chang, et al.Level and Value of Circulating Endothelial Progenitor Cells in Patients After Acute Ischemic Stroke[ J ].Stroke, 2008; 39(1): 69 - 74.
[22] Hong Zhang, Varsha Vakil, Marc Braunstein, et al.Circulating endothelial progenitor cells in multiple myeloma: implications and significance [J].Blood, 2005; 105(8): 3286-3294.
[23] Shin SJ., Jeung HC, Ahn JB., et al.Mobilized CD34~+ cells as a biomarker candidate for the efficacy of combined maximal tolerance dose and continuous infusional chemotherapy and G-CSF surge in gastric cancer[J].Cancer Lett.2008,270(2):269-270.
[24] Decai Yu, Xitai Sun, Yudong Qiu, et al.Identification and Clinical Significance of Mobilized Endothelial Progenitor Cells in Tumor Vasculogenesis of Hepatocellular Carcinoma[ J ].Clin. Cancer Res., 2007; 13(13): 3814 - 3824.
[25] RP Naik, D Jin, E Chuang, et al.Circulating endothelial progenitor cells correlate to stage in patients with invasive breast cancer [ J ].Breast Cancer Res Treat, 2008; 107(1): 133-8.
[26] Peter E Westerweel, Remco K M A C Luijten, Imo E Hoefer, et al.Haematopoietic and endothelial progenitor cells are deficient in quiescent systemic lupus erythematosus[J].Ann Rheum Dis, 2007; 66(7): 865 - 870.
[27] Ellen Lyda Burnham, Diane Sutcliffe, Michael Edwards, et al.Endothelial Progenitor Cells Are Decreased in the Circulation of Patients with Sepsis [ J ].FASEB J, 2008; 22: 964.1.
[2] Wu DL, Ha XQ, et al.Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat myocardial ischemia models.Chinese Science Bulletin , 2003,48(3):273-276.
[3] Wu DL, Ha XQ, et al.Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat myocardial ischemia models.Chinese Science Bulletin, 2003,48(3):273-276.
[4] Ha XQ, et al.Gene therapy for pathological scar with hepatocyte growth factor mediated by recombinant adenovirus vector. Science in China, 2003,46(3):320-327.
[5] Serneels L, Dejaegere T, Craessaerts K, et al. Differential contribution of the three Aph1 genes to {gamma}-secretase activity in vivo. Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1719-24.
[6] Duarte A, Hirashima M, Benedito R, et al. Dosage-sensitive requirement for mouse D114 in artery development. Genes Dev. 2004 Oct 15;18(20):2474-8.
[7] Alva JA, Iruela-Arispe ML. Notch signaling in vascular morphogenesis. Curr Opin Hematol. 2004 Jul;11(4):278-83.
[8] Lawson ND, Scheer N, Pham VN, et,al. Notch signaling is required for arterial-venous differentiation during embryonic vascular development. Development. 2001 Oct;128(19):3675-83.
[9] Taylor KL, Henderson AM, Hughes CC. Notch activation during endothelial cell network formation in vitro targets the basic HLH transcription factor HESR-1 and downregulaties VEGFR-2/KDR expression. Microvasc Res. 2002,64(3):372-83.
[10] Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nature Med.2000, 6(3):389-395.
[11] Stella MC,Trusolino L,Pennacchietti S,et al. Negative feedback regulation of Met-dependent invasive growth by Notch. Mol Cell Biol.2005 May;25(10):3982-3996.
[12] Pires-daSilva A , Sommer RJ .The evolution of signalling pathways in animal development. Nat Rev Genet ,2003 ,4 (12) 39—49.
[13] Ha XQ, et al. Effect of human hepatocyte growth factor on promoting wound healing and preventing scar formation by adenovirus-mediated gene transfer. Chinese Medical Journal,2003,116(7): 1029-1033.
[14] Rossant J, Hirashima M. Vascular development and patterning: making the right choice. Curr Opin Genet Dev. 2003,13(4):408-12.
[15] Lawson ND, Vogel AM, Weinstein BM. sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev Cell. 2002 Jul;3(1): 127-36.
[16] Serneels L, Dejaegere T, Craessaerts K, et al. Differential contribution of the three Aph1 genes to {gamma}-secretase activity in vivo. Proc Natl Acad Sci U S A. 2005 Feb 1; 102(5): 1719-24.
[17] Duarte A, Hirashima M, Benedito R, et al. Dosage-sensitive requirement for mouse D114 in artery development. Genes Dev. 2004 Oct 15;18(20):2474-8.
[18] Alva JA, Iruela-Arispe ML. Notch signaling in vascular morphogenesis. Curr Opin Hematol. 2004 Jul;11(4):278-83.
[19] Shawber CJ, Kitajewski J. Notch function in the vasculature: insights from zebrafish, mouse and man. Bioessays. 2004 Mar;26(3):225-34.
[20]. T. Kadesch, Notch signaling: the demise of elegant simplicity.Curr Opin Genet Dev,2004, 14 (5): 506-512.
[21]. Luke T.Krebs,Yingzi Xue,Christine R.Norton,et al. Notch signaling is essential for vascular morphogenesis in mice .Genes Dev., 2000, 14(1): 1343-1352.
[22] C Mailhos, U Modlich, J Lewis, et al Delta4, an endothelial specific notch ligand expressed at sites of physiological and tumor angiogenesis.Differentiation, 2001, 69(2-3): 135-144.
[23] H Diez,A Fischer,A Winkler,et al . Hypoxia-mediated activation of D114-Notch-Hey2 signaling in endothelial progenitor cells and adoption of arterial cell fate. Exp Cell Res, 2007, 313(1): 1-9.
[24]. Liu ZJ, Shirakawa T, Li Y, et,al. Regulation of Notchl and D114 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis. Mol Cell Biol. 2003,23(1): 14-25.
[25].Scehnet JS,Jiang W,Kumar SR,et al.Inhibition of D114 mediated signaling induces proliferation of immature vessels and results in poor tissue perfusion. Blood, 2007,109:4753-4760.
[26] Hellstrom M,Phng LK,Hofmann JJ,et al.D114 signaling through Notch1 regulates formation of tip cells during angiogenesis. Nature, 2007,445(7129):776-780.
[31] Peichev M,Naiyer AJ,Pereira D, et al.Expression of VEGF-2 and AC133 by circulating human CD34~+ cells identifies a population of functional endothelial precursors[J]. Blood, 2000,95(3):952-958.
[32] Yin A,Miraglia S,Zarjani E,et al. AC133,a novel marker for human hematopoie stem and progenitor cells[J]. Blood, 1997,90:5002-5012.
[1]Iwaguro H,Asahara T.Endothelial progenitor cell culture and gene transfer[J].Methods Mol Med,2005,112:239-247.
[2]Ingram DA,Cap lice NM,Mervin C,et al.Unresolved questions,changing definitions,and novel paradigms for defining endothelial progenitor cells[J].Blood,2005,106(5):1525-1531.
[3]Fox A,Smythe J,Fisher N,et al.Mobilization of endothelial progenitor cells into the circulation[J].Anaesthesist,2007,5:42-47.
[4]Ho JW,Pang RW,Lau C,et al.Significance of circulating endothelial progenitor cells in hepatocellular carcinoma[J].Hepatology,2006,44(4):836-843.
[5]Christoph Kalka,Hassan Tehrani,Bemd Laudenberg,et al.VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease[J].Ann.Thorac.Surg.,2000;70(3):829-834.
[6]王改玲,肖传实,邱龄,等.粒细胞集落刺激因子对内皮前体细胞的动员[J].医学研究生学报,2006,19(4):3342-3371.
[7]肖刚峰,张怀勤,季亢挺,等.丹参素对体外培养内皮祖细胞数量和功能的影响[J].华西药学杂志,2006,21(3):2322-2341.
[8]Kerstin Strehlow,Nikos Werner,Jan Berweiler,et al.Estrogen Increases Bone Marrow-Derived Endothelial Progenitor Cell Production and Diminishes Neointima Formation[J].Circulation,2003;107(24):3059-3065.
[9]Askari AT,Unzek S,Popovic ZB,et al,Effect of stromal-cell-derived factor-1 on stem-cell homing and tissue regeneration in ischacmic cardiomyopathy[J].Lance,2003,362(9385):697-703.
[10]Murayama T,Tepper OM,Silver M,et al.Determination of bone marrow derived endothelial progenitor cell significance in angiogenic growth factor induced neovascularization in vivo[J].Exp Hematol,2002,30(8):967.
[11]Chavakis E,Aicher A,Heeschen C,et al.Role of β2-integrins for homing and neovascularization capacity of endothelial progenitor cells[J].Exp Med,2005,201(1):63-72.
[12]Verama S,Kuliszewski MA,Li SH,et al.C-reactive protein attenuates endothelial progenitor cell survival,differentiation,and function:further evidence of a mechanistic link between C-reactive protein and cardiovascular disease[J].Circulation,2004,109(17):2058.
[13]Urbich C,Heeschen C,Aicher A,et al.Cathepsin-L is required for endothelial progenitor cell induced neovascularization[J].Nat Med,2005,11(2):206-213.
[14]Hur J,Yoon CH,Lee CS,et al.Akt is a key modulator of endothelial progenitor cell trafficking in ischemic muscle[J].Stem Cells,2007,25(7):1769-1778.
[15]Dirk It.Walter,Kilian Rittig,Ferdinand H.Bahlmann,et al.Statin Therapy Accelerates Reendothelialization:A Novel Effect Involving Mobilization and Incorporation of Bone Marrow-Derived Endothelial Progenitor Cells [J]. Circulation,2002;105(25):3017-3024.
[16] Ying Guo, Rebecca Chan, Heather Ramsey,et al. The homeoprotein Hex is required for hemangioblast differentiation[J].Blood, 2003;102(7):2428-2435.
[17] Landmeser U , Eug berdiug N , Bahlmann FH , et al. Statin-induced improvement of endothelial progenitor cell mobilization , myocardial neovascularization , left ventricular function and survival after experimental myocardial infarction requires endothelial nitricoxide synthase[ J ].Circulation , 2004 , 110(14): 1933.
[18] Blann AD ,Pretorius A. Circulating endothelial cells and endothelial progenitor cells :Two sides of the same coin or two different coins [ J ] . Atherosclerosis , 2006 , 188 (1) :12-18.
[19] Schmidt-Lucke C, Rossig L, Fichtlscherer S, et al. Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair[ J ]. Circulation.2005;111(22):2981-2987.
[20] Gian Paolo Fadini, Saverio Sartore, Mattia Albiero, et al.Number and Function of Endothelial Progenitor Cells as a Marker of Severity for Diabetic Vasculopathy[ J ].Arterioscler. Thromb. Vasc. Biol., 2006; 26(9): 2140 - 2146.
[21] Hon-Kan Yip, Li-Teh Chang, Wen-Neng Chang, et al.Level and Value of Circulating Endothelial Progenitor Cells in Patients After Acute Ischemic Stroke[ J ].Stroke, 2008; 39(1): 69 - 74.
[22] Hong Zhang, Varsha Vakil, Marc Braunstein, et al.Circulating endothelial progenitor cells in multiple myeloma: implications and significance [J].Blood, 2005; 105(8): 3286-3294.
[23] Shin SJ., Jeung HC, Ahn JB., et al.Mobilized CD34~+ cells as a biomarker candidate for the efficacy of combined maximal tolerance dose and continuous infusional chemotherapy and G-CSF surge in gastric cancer[J].Cancer Lett.2008,270(2):269-270.
[24] Decai Yu, Xitai Sun, Yudong Qiu, et al.Identification and Clinical Significance of Mobilized Endothelial Progenitor Cells in Tumor Vasculogenesis of Hepatocellular Carcinoma[ J ].Clin. Cancer Res., 2007; 13(13): 3814 - 3824.
[25] RP Naik, D Jin, E Chuang, et al.Circulating endothelial progenitor cells correlate to stage in patients with invasive breast cancer [ J ].Breast Cancer Res Treat, 2008; 107(1): 133-8.
[26] Peter E Westerweel, Remco K M A C Luijten, Imo E Hoefer, et al.Haematopoietic and endothelial progenitor cells are deficient in quiescent systemic lupus erythematosus[J].Ann Rheum Dis, 2007; 66(7): 865 - 870.
[27] Ellen Lyda Burnham, Diane Sutcliffe, Michael Edwards, et al.Endothelial Progenitor Cells Are Decreased in the Circulation of Patients with Sepsis [ J ].FASEB J, 2008; 22: 964.1.