体外诱导小鼠心脏CD51~+细胞分化为血管内皮细胞的研究
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摘要
目的探讨小鼠心脏CD51~+细胞的分离培养及体外定向诱导分化为血管内皮细胞的可行性,为其治疗心脏血管内皮损伤提供理论依据。方法取出日龄为7 d的C57 BL/6乳鼠心脏,用流式分选法分选原代细胞、悬浮培养法培养及纯化心脏CD51~+细胞。用内皮生长培养基(EGM-2)对纯化和扩增后的CD51~+细胞进行体外诱导内皮分化20 d。诱导后观察细胞形态变化;实时荧光定量PCR分析内皮相关基因CD31、CD34、v WF、VEGFR-2及VE-Cadherin的mRNA表达;细胞免疫荧光技术、流式表型分析术检测内皮相关标志CD31、VEGFR-2及v WF的表达;测定细胞一氧化氮(NO)含量、行体外血管形成实验以鉴定其功能。结果诱导内皮分化后细胞变为短梭形,呈铺路石样排列生长;荧光定量PCR结果显示诱导后细胞CD31、CD34、v WF、VEGFR-2、VE-Cadherin的mRNA相对表达量较诱导前增加(t值分别为24.529、6.383、25.872、5.256、5.255,P均<0.05);细胞免疫荧光染色及流式表型分析结果均显示诱导后细胞表达CD31、VEGFR-2及v WF;诱导后细胞NO含量较诱导前增加(t=9.549,P=0.011)并具有体外形成管腔样结构的能力。结论心脏CD51~+细胞具有在体外定向诱导分化为血管内皮细胞的能力。
Objective To investigate the feasibility of isolation,culture and in vitro induction of the differentiation of cardiac CD51~+cells into vascular endothelial cells,aiming to provide theoretical evidence for the treatment of cardiac vascular endothelial injury. Methods The heart tissues were collected from 7-day-old C57 BL/6 mice. Primary cells were isolated by flow cytometry and cell sorting. The cardiac CD51~+cells were cultured and purified by suspension cell culture method. After cell purification and proliferation,the cardiac CD51~+cells were subjected to in vitro induction of differentiation into endothelial cells in endothelial cell growth medium-2( EGM-2) for 20 days. After in vitro induction,the changes in cell morphology were observed. The expression levels of CD31,CD34,v WF,VEGFR-2 and VE-Cadherin mRNA were quantitatively measured by real-time fluorescent quantitative PCR. The expression levels of CD31,VEGFR-2 and v WF were detected by immunofluorescent staining and flow cytometric immunophenotying. The level of nitric oxide( NO)was measured and the function of the induced endothelial cells was evaluated by angiogenesis assay in vitro.Results The morphology of cardiac CD51~+cells was changed from the long-spindle to short-spindle shape and the induced cells grew in a cobblestone-like pattern. The results of fluorescent quantitative PCR demonstrated that the relative expression levels of CD31,CD34,v WF,VEGFR-2 and VE-Cadherin mRNA were significantly up-regulated after induction( t = 24. 529,6. 383,25. 872,5. 256 and 5. 255,all P < 0. 05). Both immunofluorescent staining and flow cytometric immunophenotying revealed that the cells expressed CD31,VEGFR-2,and v WF after induction. The quantity of NO was considerably increased after induction( t = 9. 549,P =0. 011) and had the ability to form lumen-like structures in vitro. Conclusion Cardiac CD51~+cells can be induced to differentiate into vascular endothelial cells in vitro.
Objective To investigate the feasibility of isolation,culture and in vitro induction of the differentiation of cardiac CD51~+cells into vascular endothelial cells,aiming to provide theoretical evidence for the treatment of cardiac vascular endothelial injury. Methods The heart tissues were collected from 7-day-old C57 BL/6 mice. Primary cells were isolated by flow cytometry and cell sorting. The cardiac CD51~+cells were cultured and purified by suspension cell culture method. After cell purification and proliferation,the cardiac CD51~+cells were subjected to in vitro induction of differentiation into endothelial cells in endothelial cell growth medium-2( EGM-2) for 20 days. After in vitro induction,the changes in cell morphology were observed. The expression levels of CD31,CD34,v WF,VEGFR-2 and VE-Cadherin mRNA were quantitatively measured by real-time fluorescent quantitative PCR. The expression levels of CD31,VEGFR-2 and v WF were detected by immunofluorescent staining and flow cytometric immunophenotying. The level of nitric oxide( NO)was measured and the function of the induced endothelial cells was evaluated by angiogenesis assay in vitro.Results The morphology of cardiac CD51~+cells was changed from the long-spindle to short-spindle shape and the induced cells grew in a cobblestone-like pattern. The results of fluorescent quantitative PCR demonstrated that the relative expression levels of CD31,CD34,v WF,VEGFR-2 and VE-Cadherin mRNA were significantly up-regulated after induction( t = 24. 529,6. 383,25. 872,5. 256 and 5. 255,all P < 0. 05). Both immunofluorescent staining and flow cytometric immunophenotying revealed that the cells expressed CD31,VEGFR-2,and v WF after induction. The quantity of NO was considerably increased after induction( t = 9. 549,P =0. 011) and had the ability to form lumen-like structures in vitro. Conclusion Cardiac CD51~+cells can be induced to differentiate into vascular endothelial cells in vitro.
引文
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[3]Bulum J,Ernst A,Strozzi M.The impact of successful manual thrombus aspiration on in-stent restenosis after primary PCI:angiographic and clinical follow-up.Coron Artery Dis,2012,23(7):487-491.
[4]Andersen DC,Andersen P,Schneider M,Jensen HB,Sheikh SP.Murine“cardiospheres”are not a source of stem cells with cardiomyogenic potential.Stem Cells,2009,27(7):1571-1581.
[5]陈阳,李桂兰,姜美花,彭朝权.小鼠心源性CD51阳性细胞的分离与鉴定.中山大学学报(医学科学版),2014,35(3):424-430.
[6]林婉文,杨珂,廖延,姜美花,彭朝权.CD51阳性心肌细胞对心肌梗死后小鼠心功能改善的作用.新医学,2015,46(6):350-355.
[7]王正东,李平,林智海,甘剑挺.血管损伤及PCI术后再狭窄机制的研究进展和相应对策.医学综述,2016,22(2):280-283.
[8]Kwon JS,Kim YS,Cho AS,Kim JS,Jeong SY,Hong MH,Jeong MH,Ahn Y.Origin of restenosis after drug-eluting stent implantation in hyperglycemia is inflammatory cells and thrombus.J Atheroscler Thromb,2011,18(7):604-615.
[9]Perkins LE.Preclinical models of restenosis and their application in the evaluation of drug-eluting stent systems.Vet Pathol,2010,47(1):58-76.
[10]Guerra E,Byrne RA,Kastrati A.Pharmacological inhibition of coronary restenosis:systemic and local approaches.Expert Opin Pharmacother,2014,15(15):2155-2171.
[11]Lupi A,Secco GG,Rognoni A,Rossi L,Lazzero M,Nardi F,Rolla R,Bellomo G,Bongo AS,Di Mario C.Plasma fibrinogen levels and restenosis after primary percutaneous coronary intervention.J Thromb Thrombolysis,2012,33(4):308-317.
[12]Gutman D,Golomb G.Liposomal alendronate for the treatment of restenosis.J Control Release,2012,161(2):619-627.
[13]Chaabane C,Otsuka F,Virmani R,Bochaton-Piallat ML.Biological responses in stented arteries.Cardiovasc Res,2013,99(2):353-363.
[14]Cassese S,Byrne RA,Tada T,Pinieck S,Joner M,Ibrahim T,King LA,Fusaro M,Laugwitz KL,Kastrati A.Incidence and predictors of restenosis after coronary stenting in 10 004 patients with surveillance angiography.Heart,2014,100(2):153-159.
[15]沈根,张顺,叶红华.他汀类药物对内皮祖细胞作用的研究进展.新医学,2015,46(12):789-793.
[16]孙文博,王贺,司春婴,解金红,陈玉善,柴爽爽,关怀敏.内皮祖细胞对药物洗脱支架置入术后再狭窄及再内皮化影响的研究进展.中国医药导报,2016,13(24):54-57.
[17]Liao J,Chen X,Li Y,Ge Z,Duan H,Zou Y,Ge J.Transfer of bone-marrow-derived mesenchymal stem cells influences vascular remodeling and calcification after balloon injury in hyperlipidemic rats.J Biomed Biotechnol,2012,2012:165296.
[18]Fuchs E,Tumbar T,Guasch G.Socializing with the neighbors:stem cells and their niche.Cell,2004,116(6):769-778.
[19]Beltrami AP,Barlucchi L,Torella D,Baker M,Limana F,Chimenti S,Kasahara H,Rota M,Musso E,Urbanek K,Leri A,Kajstura J,Nadal-Ginard B,Anversa P.Adult cardiac stem cells are multipotent and support myocardial regeneration.Cell,2003,114(6):763-776.
[20]Sheppard D.Roles of alphav integrins in vascular biology and pulmonary pathology.Curr Opin Cell Biol,2004,16(5):552-557.
[21]Kuwana M,Okazaki Y,Kodama H,Satoh T,Kawakami Y,Ikeda Y.Endothelial differentiation potential of human monocyte-derived multipotential cells.Stem Cells,2006,24(12):2733-2743.
[22]胡庆松,聂如琼.血管内皮细胞标志物研究进展.岭南心血管病杂志,2012,18(2):196-201.
[23]Vestweber D.VE-cadherin:the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation.Arterioscler Thromb Vasc Biol,2008,28(2):223-232.