冻存脐带血中内皮祖细胞的分离培养及生物学特性鉴定
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摘要
目的:建立和开发一种新型的从冻存脐带血中分离培养内皮祖细胞(EPC)的方法,探讨其生物学特性,提高冻存脐带血分离获得EPC的成功率。方法:自液氮罐中收集公共库中2000年至2001年保存的12例冻存脐带血,37℃水浴锅中快速复苏后,磷酸缓冲盐溶液(PBS缓冲液)洗涤,获得总有核细胞(TNCs),将TNCs接种至培养皿中诱导扩增EPC。使用流式细胞术和免疫荧光方法鉴定EPC并确定纯度。采用下列方法体外鉴定内皮祖细胞的功能:荧光显微镜观察细胞特异性摄取Dil-Ac-LDL和FITC-UEA-I的功能、在基质胶中形成毛细管样结构的情况,ELISA法检测释放VEGF因子的功能。结果:培养3周后,出现1-5个鹅卵石铺路石样内皮祖细胞集落。体外培养第1-3代EPC,其表面CD31、CD34、CD144和VEGFR (CD309)标记阳性率分别为(92. 91±5. 20)%、(30. 0±23. 27)%、(88. 55±3. 83)%和(67. 21±12. 12)%。EPC细胞具有特异性内吞Dil-Ac-LDL和FITCUEA-I、形成毛细管结构,释放低浓度VEGF因子等功能。结论:采用本方法从冻存脐带血中分离EPC的稳定性与重复性高,从冻存的脐带血中分离EPC的成功率可提高至85%。该方法为临床应用丰富的内皮祖细胞奠定了基础。
Objective: To establish a novel method to isolate endothelial progenitor cells( EPC) from cryopreserved umbilical cord blood( cryoUCB),to investigate the biological characteristics of EPC and to improve the rate of EPC obtained from cryoUCB. Methods: Tw elve cryoUCB samples during 2000 to 2001 years w ere collected from allogeneic cord blood bank,cryoUCB w as thaw ed rapidly in a w ater bath at 37 ℃,total nucleated cells( TNCs) w ere w ashed by phosphate-buffered saline( PBS). TNCs w ere seeded onto fibronectin-coated dishes to isolate EPC. Flow cytometry and immunofluorescence w ere used to identify EPC. The function of EPC w as identified in vitro, such as the incorporation of Dil-Ac-LDL and FITC-UEA-I,the formation of capillary-like structure in matrigel,and the release of VEGF by ELISA. Results: One to five cluster of cobble stone-like cells appeared at 2-3 w eeks after seeding.Flow cytometric analysis show ed that positive rates of CD31,CD34,CD144,and VEGFR( CD309) w ere( 92. 91 ±5. 20) %,( 30. 0 ± 23. 27) %,( 88. 55 ± 3. 83) % and( 67. 21 ± 12. 12) % in passage 1 to passage 3 of EPC. EPC could uptake Dil-Ac-LDL and FITC-UEA-I,form capillary-like netw ork on M atriget and release VEGF. Conclusion:EPC had been successfully isolated from cryopreserved umbilical cord blood by this method w ith high stability and reproducibility. EPC can be obtained in 85% frozen umbilical cord blood. This method may lay a foundation to supply abundant EPC for clinical application.
Objective: To establish a novel method to isolate endothelial progenitor cells( EPC) from cryopreserved umbilical cord blood( cryoUCB),to investigate the biological characteristics of EPC and to improve the rate of EPC obtained from cryoUCB. Methods: Tw elve cryoUCB samples during 2000 to 2001 years w ere collected from allogeneic cord blood bank,cryoUCB w as thaw ed rapidly in a w ater bath at 37 ℃,total nucleated cells( TNCs) w ere w ashed by phosphate-buffered saline( PBS). TNCs w ere seeded onto fibronectin-coated dishes to isolate EPC. Flow cytometry and immunofluorescence w ere used to identify EPC. The function of EPC w as identified in vitro, such as the incorporation of Dil-Ac-LDL and FITC-UEA-I,the formation of capillary-like structure in matrigel,and the release of VEGF by ELISA. Results: One to five cluster of cobble stone-like cells appeared at 2-3 w eeks after seeding.Flow cytometric analysis show ed that positive rates of CD31,CD34,CD144,and VEGFR( CD309) w ere( 92. 91 ±5. 20) %,( 30. 0 ± 23. 27) %,( 88. 55 ± 3. 83) % and( 67. 21 ± 12. 12) % in passage 1 to passage 3 of EPC. EPC could uptake Dil-Ac-LDL and FITC-UEA-I,form capillary-like netw ork on M atriget and release VEGF. Conclusion:EPC had been successfully isolated from cryopreserved umbilical cord blood by this method w ith high stability and reproducibility. EPC can be obtained in 85% frozen umbilical cord blood. This method may lay a foundation to supply abundant EPC for clinical application.
引文
1 Werner N,Kosiol S,Schiegl T,et al.Circulating endothelial progenitor cells and cardiovascular outcomes.N Engl J Med,2005;10(353):999-1007.
2 Hill JM,Zalos G,Halcox JP,et al.Circulating endothelial progenitor cells,vascular function,and cardiovascular risk.N Engl J Med,2003;348(7):593-600.
3 Rosenzweig A.Endothelial progenitor cells.N Engl J Med,2003;348(7):581-582.
4 Kim H,Kim S,Baek SH,et al.Pivotal cytoprotective mediators and promising therapeutic strategies for endothelial progenitor cell-based cardiovascular regeneration.Stem Cells Int,2016;2016(3):1-14.
5 Au P,Daheron LM,Duda DG,et al.Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels.Blood,2008;111(3):1302-1305.
6 Kawamoto A,Murayama T,Kusano K,et al.Synergistic effect of bone marrow mobilization and vascular endothelial growth factor-2gene therapy in myocardial ischemia.Circulation,2004;110(11):1398-1405.
7 Schachinger V,Assmus B,Britten MB,et al.Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction:final one-year results of the TOPCARE-AMI Trial.J Am Coll Cardiol,2004;44(8):1690-1699.
8 Assmus B,Sch?chinger V,Teupe C,et al.Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction(TOPCARE-AMI).Circulation,2002;106(24):3009-3017.
9 Asahara T,Murohara T,Sullivan A,et al.Isolation of putative progenitor endothelial cells for angiogenesis.Science,1997;5302(275):964-967.
10 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.Circulation,2005;111(22):2981-2987.
11 Vasa M,Fichtlscherer S,Aicher A,et al.Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease.Circ Res,2001;89(1):E1-7.
12 Fadini GP,Coracina A,Baesso I,et al.Peripheral blood CD34+KDR+endothelial progenitor cells are determinants of subclinical atherosclerosis in a middle-aged general population.Stroke,2006;37(9):2277-2282.
13姚根宏,栾建凤.内皮祖细胞生物学特性及其临床应用研究进展.中国实验血液学杂志,2008;16(06):1473-1476.
14 Shirota T,Yasui H,Shimokawa H,et al.Fabrication of endothelial progenitor cell(EPC)-seeded intravascular stent devices and in vitro endothelialization on hybrid vascular tissue.Biomaterials,2003;24(13):2295-2302.
15唐欲博,陈孝,黄保丁,等.人骨髓来源内皮祖细胞的分离培养及生物学特性的鉴定.中国骨科临床与基础研究杂志,2009;1(01):56-61.
16辛毅,刘小希,赵伟,等.差速贴壁法分离兔骨髓源性间充质干细胞和内皮祖细胞及其生物学特性的研究.中国实验血液学杂志,2013;21(3):746-753.
17 Jang JH,Kim SK,Choi JE,et al.Endothelial progenitor cell differentiation using cryopreserved,umbilical cord blood-derived mononuclear cells.Acta Pharmacol Sin,2007;28(3):367-374.
18 Vanneaux V,El-Ayoubi F,Delmau C,et al.In vitro and in vivo analysis of endothelial progenitor cells from cryopreserved umbilical cord blood:are we ready for clinical application?Cell transplant,2010;19(9):1143-1155.
19方立建,宋鄂,栾瑛,等.人脐带血内皮祖细胞体外培养和鉴定.中国组织工程研究与临床康复,2010;14(45):8450-8454.
20 Lu X,Proctor SJ,Dickinson AM.The effect of cryopreservation on umbilical cord blood endothelial progenitor cell differentiation.Cell Transplant,2008;17(12):1423-1428.
21 Hur J,Yoon CH,Kim HS,et al.Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis.Arterioscler Thromb Vasc Biol,2004;24(2):288-293.
2 Hill JM,Zalos G,Halcox JP,et al.Circulating endothelial progenitor cells,vascular function,and cardiovascular risk.N Engl J Med,2003;348(7):593-600.
3 Rosenzweig A.Endothelial progenitor cells.N Engl J Med,2003;348(7):581-582.
4 Kim H,Kim S,Baek SH,et al.Pivotal cytoprotective mediators and promising therapeutic strategies for endothelial progenitor cell-based cardiovascular regeneration.Stem Cells Int,2016;2016(3):1-14.
5 Au P,Daheron LM,Duda DG,et al.Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels.Blood,2008;111(3):1302-1305.
6 Kawamoto A,Murayama T,Kusano K,et al.Synergistic effect of bone marrow mobilization and vascular endothelial growth factor-2gene therapy in myocardial ischemia.Circulation,2004;110(11):1398-1405.
7 Schachinger V,Assmus B,Britten MB,et al.Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction:final one-year results of the TOPCARE-AMI Trial.J Am Coll Cardiol,2004;44(8):1690-1699.
8 Assmus B,Sch?chinger V,Teupe C,et al.Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction(TOPCARE-AMI).Circulation,2002;106(24):3009-3017.
9 Asahara T,Murohara T,Sullivan A,et al.Isolation of putative progenitor endothelial cells for angiogenesis.Science,1997;5302(275):964-967.
10 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.Circulation,2005;111(22):2981-2987.
11 Vasa M,Fichtlscherer S,Aicher A,et al.Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease.Circ Res,2001;89(1):E1-7.
12 Fadini GP,Coracina A,Baesso I,et al.Peripheral blood CD34+KDR+endothelial progenitor cells are determinants of subclinical atherosclerosis in a middle-aged general population.Stroke,2006;37(9):2277-2282.
13姚根宏,栾建凤.内皮祖细胞生物学特性及其临床应用研究进展.中国实验血液学杂志,2008;16(06):1473-1476.
14 Shirota T,Yasui H,Shimokawa H,et al.Fabrication of endothelial progenitor cell(EPC)-seeded intravascular stent devices and in vitro endothelialization on hybrid vascular tissue.Biomaterials,2003;24(13):2295-2302.
15唐欲博,陈孝,黄保丁,等.人骨髓来源内皮祖细胞的分离培养及生物学特性的鉴定.中国骨科临床与基础研究杂志,2009;1(01):56-61.
16辛毅,刘小希,赵伟,等.差速贴壁法分离兔骨髓源性间充质干细胞和内皮祖细胞及其生物学特性的研究.中国实验血液学杂志,2013;21(3):746-753.
17 Jang JH,Kim SK,Choi JE,et al.Endothelial progenitor cell differentiation using cryopreserved,umbilical cord blood-derived mononuclear cells.Acta Pharmacol Sin,2007;28(3):367-374.
18 Vanneaux V,El-Ayoubi F,Delmau C,et al.In vitro and in vivo analysis of endothelial progenitor cells from cryopreserved umbilical cord blood:are we ready for clinical application?Cell transplant,2010;19(9):1143-1155.
19方立建,宋鄂,栾瑛,等.人脐带血内皮祖细胞体外培养和鉴定.中国组织工程研究与临床康复,2010;14(45):8450-8454.
20 Lu X,Proctor SJ,Dickinson AM.The effect of cryopreservation on umbilical cord blood endothelial progenitor cell differentiation.Cell Transplant,2008;17(12):1423-1428.
21 Hur J,Yoon CH,Kim HS,et al.Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis.Arterioscler Thromb Vasc Biol,2004;24(2):288-293.