巴曲酶对体外培养下内皮祖细胞的数量和功能的影响
摘要
目的:
血管内皮祖细胞(Endothelial progenitor cells,EPCs)是血管内皮的前体细胞,它们不仅参与胚胎期的血管发育,也存在于成年机体的骨髓及外周血,在成体血管新生中起重要作用。近年来的研究表明EPCs在下肢缺血性疾病的发生、发展中扮演了重要角色,血管内皮祖细胞数量减少预示血管内皮修复能力降低,血管疾病发生率增高。巴曲酶(Batroxobin,DF-521)是一种由蝮蛇毒液中提纯,精制而出的丝氨酸蛋白,目前临床上广泛用于包括脑梗塞、慢性下肢动脉硬化性闭塞症(ASO)在内的各种缺血性疾病的治疗,体现出其可能对于血管内皮的功能障碍有保护作用。内皮的损伤与修复之间的动态平衡是维持其正常功能的关键,EPCs是参与修复的重要因素。考虑DF-521可能通过影响EPCs促进内皮功能的改善,本文经试验观察其对于外周血EPCs数量、增殖、迁移和粘附能力的影响,旨在进一步探讨DF-521的作用机制,并为体外扩增EPCs寻找可能的诱导剂,从而提供更充足并良好的血管组织工程细胞。
方法:
实验血液样本来自健康志愿者,用于对照组和药物干预组的EPCs数量及功能检测。采用密度梯度离心法从外周血获得单个核细胞,将其接种在人纤维连接蛋白包被的培养板上。DMEM培养基培养4d,用PBS洗掉非贴壁细胞,换培养液继续培养至7d,用PBS洗掉非贴壁细胞,贴壁细胞供实验用。激光共聚焦显微镜鉴定FITC—UEA—Ⅰ和Dil—acLDL双染色阳性细胞为正在分化的EPCs;贴壁细胞随机分成4组,培养24h后干预组分别加入不同浓度的巴曲酶(0.05BU·ml~(-1)、0.1BU·ml~(-1)、0.2BU·ml~(-1))培养24h,再将其在倒置荧光显微镜下计数。分别采用MTT比色法、改良的Boyden小室和黏附能力测定实验来观察EPCs的增殖能力、迁移能力和黏附能力,再根据结果选择差异较显著的剂量再进行DF-521对EPCs数量及功能影响的时效关系实验。
结果:
1.EPCs的鉴定
分离获得的单个核细胞培养7d后形成了梭形的内皮样细胞。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,FITC-UEA-Ⅰ和Dil-acLDL双染色阳性细胞为正在分化的EPCs。
2.巴曲酶对外周血EPCs数量的影响
不同浓度的巴曲酶与EPCs培养24h均能增加其数量,其中以0.1BU·ml~(-1)在培养24h时最为显著。
3.巴曲酶对外周血EPCs增殖功能的影响
采用MTT比色法检测巴曲酶对EPCS增殖能力的影响,结果显示:不同浓度的巴曲酶均显著增加EPCs的增殖能力,其中以0.1BU·ml~(-1)在培养24h时最为显著。
4.巴曲酶对外周血EPCs迁移功能的影响
采用改良的Boydne小室检测巴曲酶对EPCs迁移能力的影响,在200倍显微镜下计数迁移的细胞。巴曲酶明显提高EPCs的迁移能力,其中以0.1BU·ml~(-1)在培养24h时最为显著。
5.巴曲酶对外周血EPCs黏附功能的影响
为了观察巴曲酶对外周血EPCs豁附能力的影响,先将不同浓度的巴曲酶与EPCs培养24h,然后将相同数量的EPCs重新接种到包被有人纤维连接蛋白的培养板上培养30h,结果显示0.05 BU·ml~(-1)的巴曲酶明显提高EPCs的黏附能力,且在培养24h时最为显著。
结论:
1.巴曲酶可以增加外周血EPCs数量,提高EPCs的增殖能力、迁移能力和黏附能力。
2.巴曲酶对EPCs数量和功能的影响呈一定的时间依赖关系;
3.可以从外周血中的单个核细胞分离和培养血管内皮祖细胞。
Objective:
Vascular endothelial progenitor cells are the processor of endothelial cells.Increasing evidence suggests that circulating progenitor cells contribute to postnatal neovascularization.These cells home to site of ischemia,adopt an endothelial phenotype,and contribute to new blood vessel formation,the identity of the circulating cells that contribute to neovascularization is not entirely clear.Bone-marrow derived hematopoietic progenitor cells can give rise to endothelial progenitor cells and contribute to endothelial recovery and new capillary formation after ischemia.Batroxobin(DF-521) is widely used in the Primary and secondary Prevention of vascular diseases as a kind of serine stretch protein purified from venine of bothrops moojeni whichi show DF-521 may contribute to the protection of functional disturbance of vascular endothelium.The kinesis balance of impairment and repairment of endothelium is the key to maintan the normal funtion.Considering DF-521 may improve the function of endothelium by impact EPCs,we investigated whether DF-521 augments the number of EPCs) and promotes EPCs proliferation、migration and adhesion.
Methods:
Total monoclear cells(MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation,and then the cells were plated on fibronectin-coated culture dishes.After being cultured for 7d,the attached cells were stimulated with DF-521(final concentration:0.05,0.1,0.2 BU·ml~(-1)) for the respective time points(6,12,24 and 48h).EPCs were characterized as adherent cells double positive for DiI-LDL-uptake and lectin binding by direct flurescent staining under a laser scanning confocal microscope.EPCs were further documented by demonstrating the expression of CD34,VEGFR-2,CD133 with flow cytometry.Proliferation and migration of EPCs were assayed by MTT assay,modified Boyden chamber assay respectively.EPCs adhesion assay was performed by replating it on fibronectin-coated dishes and the adherent cells were then counted.
Results:
Incubation of isolated human MNCs with DF-521 dose- and time-dependently increased the number of EPCs,while reached the maximum 24h after the administration at 0.1 BU·ml~(-1)(1-foldincrease,P＜0.05).In addition,DF-521 also promoted proliferation,migration and adhesion of EPCs.
Conclusion:
1.The result of the present study define a novel mechanism of the action of DF-521:the augment of EPCs with enhaced functional activity concluding proliferation、migration and adhesion in vitro.
2.DF-521 could enhace the functional activity concluding proliferation、migration and adhesion of EPCs in vitro in particularly time-course relationship.
3.EPCs can be isolated and cultured from monoclear cells from peripheral blood.
血管内皮祖细胞(Endothelial progenitor cells,EPCs)是血管内皮的前体细胞,它们不仅参与胚胎期的血管发育,也存在于成年机体的骨髓及外周血,在成体血管新生中起重要作用。近年来的研究表明EPCs在下肢缺血性疾病的发生、发展中扮演了重要角色,血管内皮祖细胞数量减少预示血管内皮修复能力降低,血管疾病发生率增高。巴曲酶(Batroxobin,DF-521)是一种由蝮蛇毒液中提纯,精制而出的丝氨酸蛋白,目前临床上广泛用于包括脑梗塞、慢性下肢动脉硬化性闭塞症(ASO)在内的各种缺血性疾病的治疗,体现出其可能对于血管内皮的功能障碍有保护作用。内皮的损伤与修复之间的动态平衡是维持其正常功能的关键,EPCs是参与修复的重要因素。考虑DF-521可能通过影响EPCs促进内皮功能的改善,本文经试验观察其对于外周血EPCs数量、增殖、迁移和粘附能力的影响,旨在进一步探讨DF-521的作用机制,并为体外扩增EPCs寻找可能的诱导剂,从而提供更充足并良好的血管组织工程细胞。
方法:
实验血液样本来自健康志愿者,用于对照组和药物干预组的EPCs数量及功能检测。采用密度梯度离心法从外周血获得单个核细胞,将其接种在人纤维连接蛋白包被的培养板上。DMEM培养基培养4d,用PBS洗掉非贴壁细胞,换培养液继续培养至7d,用PBS洗掉非贴壁细胞,贴壁细胞供实验用。激光共聚焦显微镜鉴定FITC—UEA—Ⅰ和Dil—acLDL双染色阳性细胞为正在分化的EPCs;贴壁细胞随机分成4组,培养24h后干预组分别加入不同浓度的巴曲酶(0.05BU·ml~(-1)、0.1BU·ml~(-1)、0.2BU·ml~(-1))培养24h,再将其在倒置荧光显微镜下计数。分别采用MTT比色法、改良的Boyden小室和黏附能力测定实验来观察EPCs的增殖能力、迁移能力和黏附能力,再根据结果选择差异较显著的剂量再进行DF-521对EPCs数量及功能影响的时效关系实验。
结果:
1.EPCs的鉴定
分离获得的单个核细胞培养7d后形成了梭形的内皮样细胞。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,FITC-UEA-Ⅰ和Dil-acLDL双染色阳性细胞为正在分化的EPCs。
2.巴曲酶对外周血EPCs数量的影响
不同浓度的巴曲酶与EPCs培养24h均能增加其数量,其中以0.1BU·ml~(-1)在培养24h时最为显著。
3.巴曲酶对外周血EPCs增殖功能的影响
采用MTT比色法检测巴曲酶对EPCS增殖能力的影响,结果显示:不同浓度的巴曲酶均显著增加EPCs的增殖能力,其中以0.1BU·ml~(-1)在培养24h时最为显著。
4.巴曲酶对外周血EPCs迁移功能的影响
采用改良的Boydne小室检测巴曲酶对EPCs迁移能力的影响,在200倍显微镜下计数迁移的细胞。巴曲酶明显提高EPCs的迁移能力,其中以0.1BU·ml~(-1)在培养24h时最为显著。
5.巴曲酶对外周血EPCs黏附功能的影响
为了观察巴曲酶对外周血EPCs豁附能力的影响,先将不同浓度的巴曲酶与EPCs培养24h,然后将相同数量的EPCs重新接种到包被有人纤维连接蛋白的培养板上培养30h,结果显示0.05 BU·ml~(-1)的巴曲酶明显提高EPCs的黏附能力,且在培养24h时最为显著。
结论:
1.巴曲酶可以增加外周血EPCs数量,提高EPCs的增殖能力、迁移能力和黏附能力。
2.巴曲酶对EPCs数量和功能的影响呈一定的时间依赖关系;
3.可以从外周血中的单个核细胞分离和培养血管内皮祖细胞。
Objective:
Vascular endothelial progenitor cells are the processor of endothelial cells.Increasing evidence suggests that circulating progenitor cells contribute to postnatal neovascularization.These cells home to site of ischemia,adopt an endothelial phenotype,and contribute to new blood vessel formation,the identity of the circulating cells that contribute to neovascularization is not entirely clear.Bone-marrow derived hematopoietic progenitor cells can give rise to endothelial progenitor cells and contribute to endothelial recovery and new capillary formation after ischemia.Batroxobin(DF-521) is widely used in the Primary and secondary Prevention of vascular diseases as a kind of serine stretch protein purified from venine of bothrops moojeni whichi show DF-521 may contribute to the protection of functional disturbance of vascular endothelium.The kinesis balance of impairment and repairment of endothelium is the key to maintan the normal funtion.Considering DF-521 may improve the function of endothelium by impact EPCs,we investigated whether DF-521 augments the number of EPCs) and promotes EPCs proliferation、migration and adhesion.
Methods:
Total monoclear cells(MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation,and then the cells were plated on fibronectin-coated culture dishes.After being cultured for 7d,the attached cells were stimulated with DF-521(final concentration:0.05,0.1,0.2 BU·ml~(-1)) for the respective time points(6,12,24 and 48h).EPCs were characterized as adherent cells double positive for DiI-LDL-uptake and lectin binding by direct flurescent staining under a laser scanning confocal microscope.EPCs were further documented by demonstrating the expression of CD34,VEGFR-2,CD133 with flow cytometry.Proliferation and migration of EPCs were assayed by MTT assay,modified Boyden chamber assay respectively.EPCs adhesion assay was performed by replating it on fibronectin-coated dishes and the adherent cells were then counted.
Results:
Incubation of isolated human MNCs with DF-521 dose- and time-dependently increased the number of EPCs,while reached the maximum 24h after the administration at 0.1 BU·ml~(-1)(1-foldincrease,P＜0.05).In addition,DF-521 also promoted proliferation,migration and adhesion of EPCs.
Conclusion:
1.The result of the present study define a novel mechanism of the action of DF-521:the augment of EPCs with enhaced functional activity concluding proliferation、migration and adhesion in vitro.
2.DF-521 could enhace the functional activity concluding proliferation、migration and adhesion of EPCs in vitro in particularly time-course relationship.
3.EPCs can be isolated and cultured from monoclear cells from peripheral blood.
引文
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2 Asahara T,Murohara T,Sullivan A et al.1997.Isolation of endothelial progenitor cells for angiogenesis[J].Science,275:964-967.
3 Invernici G,Emanueli C,Madeddu P,et al.Human fetal aorta contains vascular progenitor cells capable of inducing vasculogenesis,angiogenesis,and myogenesis in vitro and in a murine model of peripheral ischemia[J].Am J Pathol,2007,170(6):1879-1892.
4 Stephanie Cherqui,Sunil M.Kurian,Olivier Schussler,et al.Isolation and Angiogenesis by Endothelial Progenitors in the Fetal Liver[J].Stem Cells,2006,24:44-54.
5 Peichev M,Naiyer A J,Pereira D,et al.Expression of VEGFR-2 and AC133 by circulating human CD341 cells identifies a population of functional endothelial precursors [J] . Blood, 2000, 95:952-958.
6 Gehling U M, Ergun S, Schumatcher U, et al. In vitro differentiation of endothelial cells from AC133-positive progenitor cells [J] .Blood,2000,95:3106-3112.
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1 Hill JM,Zalos G,Halcox JP,et al.Circulating endothelial progenitor cells,vascular function,and cardiovascular risk[J].N Engl J Med,2003,348:593-600.
2 Asahara T,Murohara T,Sullivan A et al.1997.Isolation of endothelial progenitor cells for angiogenesis[J].Science,275:964-967.
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4 Stephanie Cherqui,Sunil M.Kurian,Olivier Schussler,et al.Isolation and Angiogenesis by Endothelial Progenitors in the Fetal Liver[J].Stem Cells,2006,24:44-54.
5 Peichev M,Naiyer A J,Pereira D,et al.Expression of VEGFR-2 and AC133 by circulating human CD341 cells identifies a population of functional endothelial precursors [J] . Blood, 2000, 95:952-958.
6 Gehling U M, Ergun S, Schumatcher U, et al. In vitro differentiation of endothelial cells from AC133-positive progenitor cells [J] .Blood,2000,95:3106-3112.
7 Erik B. Friedrich, Katrin Walenta, John Scharlau, et al.CD34 -/CD133+/VEGFR-2+ Endothelial Progenitor Cell Subpopulation With Potent Vasoregenerative Capacities [J] .Cirl Res, 2006, 98:20-25.
8 Choi K.Kennedy M, Kazarov A, et al. A common precursor for hematopoietic and endothelial cells [J] . Development, 1998,125:725-732.
9 Eric Ackah, Jun Yu, Stefan Zoellner, et al. Akt1/protein kinase Bα is critical for ischemic and VEGF-mediated angiogenesis [J] . J Clin Invest, 2005,115:2119-2127.
10 Hattori K, Dias S,Heissig B, et al. Vascular Endothelial Growth Factor and Angiopoietin-1 Stimulate Postnatal Hematopoiesis by Recruitment of Vasculogenic and Hematopoietic Stem Cells [J] . J Exp Med,2001,193:1005-1014.
11 Atluri P, Liao GP, Panlilio CM, et al. Neovasculogenic therapy to augment perfusion and preserve viability in ischemic cardiomyopathy [J] . Ann Thorac Surg, 2006, 81(5):1728-1736.
12 Zheng JH, Pyatt DW, Gross SA,et al. Hydroquinone modulates the GM-CSF signaling pathway in TF-1 cells [J] . Leukemia, 2004 ,18(7):1296-1304.
13 Wolfram O, Jentsch-Ullrich K,Wagner A,et al. G-CSF-Induced Mobilization of CD34+ Progenitor Cells and Proarrhythmic Effects in Patients with Severe Coronary Artery Disease [J] . PACE 2007;30:S166-S169.
14 Ohiki Y,Heissig B, Sato Y, et al. Granulocyte ony-stimulating factor promotes neovascularization by releasing vascular endothelial growth factor from neutrophils [J] . FASEB J,2005 ,19(14): 2005-2007.
15 Heeschen C, Aicher A, Lehmann R, et al. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization [J] . Blood, 2003,102:1340-1346.
16 Bahlmann FH, De Groot K, Spandau JM, et al. Erythropoietin regulates endothelial progenitor cells [J] . Blood, 2004,103:921-926.
17 Hirata A, Minamino T, Asanuma H, et al. Erythropoietin Enhances Neovascularization of Ischemic Myocardium and Improves Left Ventricular Dysfunction After Myocardial Infarction in Dogs [J] .J Am Coll Cardiol, 2006, 48(1):176-184.
18 George J, Goldstein E, Abashidze A, et al. Erythropoietin promotes endothelial progenitor cell proliferative and adhesive properties in a PI 3-kinase-dependent manner [J] . Cardiovascular Research,2005, 68(2): 299-230.