1种新来源的内皮祖细胞提取分离方法
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摘要
目的探索1种新来源内皮祖细胞(endothelial progenitor cells,EPCs)的提取分离方法,以增加EPCs细胞数量、提高增殖活性。方法拟从胎鼠肺提取分离培养EPCs,分别从形态学、表面标志物、吞噬功能和体外管腔形成等方面验证所提取细胞为EPCs。同时将其与骨髓中提取的内皮祖细胞的细胞增殖活力进行比较。结果从胎鼠肺提取分离培养的细胞,其形态特征与EPCs一致,细胞表达CD31、CD34、CD133、VEGFR2等EPCs特异标志物,可同时吞噬Dil-ac-LDL和FITC-UEA-1;体外可形成管腔样结构,功能上符合EPCs的表现。胎鼠肺提取的细胞增殖活力明显高于传统从骨髓提取的EPCs。结论胎鼠肺中可提取出数量和质量较好的EPCs,为进一步实验提供可靠的细胞来源。
To explore a new method for the extraction and isolation of endothelial progenitor cells(EPCs) and increase the number of EPCs and the proliferative activity, we isolated EPCs from fetal rat lung and verified these cells with morphology, cell surface markers, phagocytosis and blood vessels formation. The proliferation activity of the isolated EPCs was compared with that of EPCs extracted from bone marrow. the cells isolated from fetal rat lung demonstrated the same morphological characteristics with EPCs, and expressed the specific markers of EPCs, such as CD31, CD34, CD133, VEGFR2 and so on. Also, these cells could engulf Dil-ac-LDL and FITC-UEA-1 and form lumen structure in vitro. All in a word, these cells extracted from fetal rat lung were authenticated to be EPCs.The proliferative activity of EPCs extracted from fetal rat lung was significantly higher than that from bone marrow.So it is feasible to extract EPCs from fetal rat lung, and to provide a reliable cell source for further experiments.
To explore a new method for the extraction and isolation of endothelial progenitor cells(EPCs) and increase the number of EPCs and the proliferative activity, we isolated EPCs from fetal rat lung and verified these cells with morphology, cell surface markers, phagocytosis and blood vessels formation. The proliferation activity of the isolated EPCs was compared with that of EPCs extracted from bone marrow. the cells isolated from fetal rat lung demonstrated the same morphological characteristics with EPCs, and expressed the specific markers of EPCs, such as CD31, CD34, CD133, VEGFR2 and so on. Also, these cells could engulf Dil-ac-LDL and FITC-UEA-1 and form lumen structure in vitro. All in a word, these cells extracted from fetal rat lung were authenticated to be EPCs.The proliferative activity of EPCs extracted from fetal rat lung was significantly higher than that from bone marrow.So it is feasible to extract EPCs from fetal rat lung, and to provide a reliable cell source for further experiments.
引文
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[3]Su Y,Gao L,Teng L,et al.Id1 enhances human ovarian cancer endothelial progenitor cell angiogenesis via PI3K/Akt and NF-κB/MMP-2 signaling pathways[J].J Transl Med,2013,11(1):1-8.
[4]Ghajar CM,Peinado H,Mori H,et al.The perivascular niche regulates breast tumour dormancy[J].Nat Cell Biol,2013,15(7):807-817.
[5]陈红伟,周姝,李新伟.LPS引起的炎症状态下免疫分子A20降低人脐静脉内皮细胞损伤及机制研究[J].免疫学杂志,2018,34(2):115-121.
[6]Ferrante A,Guggino G,Di Liberto D,et al.Endothelial progenitor cells:Are they displaying a function in autoimmune disorders?[J].Mech Ageing Dev,2016,159:44-48.
[7]Rodríguez Carrio J,López P,Suárez A.Endothelial progenitor cells as mediators of the crosstalk between vascular repair and immunity:Lessons from systemic autoimmune diseases[J].Curr Med Chem,2018,25(35):4478-4496.
[8]Asahara T,Murohara T,Alison S,et al.Isolation of putative prognitor endothelial progenitor endothelial cells of angiogenesis[J].Science,1997,275(5302):964-967.
[9]Goki M,Sachiko MT,Toshiharu S,et al.First evidence that bone marrow cells contribute to the construction of tissueengineered vascular autografts in vivo[J].Circulation,2003,108(14):1729-1734.
[10]Simper D,Stalboerger PG,Paneta CJ,et al.Smooth muscle progenitor cells in human blood[J].Circulation,2002,106(10):1199-1204.
[11]Lin Y,Weisdorf DJ,Solovey A,et al.Origins of circulating endothelial cells and endothelial out growth from blood[J].ClinInvest,2000,105(1):71-77.
[12]Yoon Y,Andrea W,Lindsay H,et al.Clonaly expanded novel multipotent stem cells from human bone marrow regenerate myocardiam after myocardial infarction[J].Clin Invest,2005,115(2):326-338.
[13]JunY,Hiroshi I,Masanofi H,et al.Flk1-positive cells derived from embryonic stem cells serve as vascular progenitor[J].Nature,2000,408(6808):92-96.
[14]Miranvile A,Heeschen C,Sengens C,et al.Improvement of postnatal neovascularization by human adipose tissue derived stem cells[J].Circulation,2004,110(3):349-355.
[15]Salvadori ML,Lessa TB,Russo FB,et al.Mice embryology:A microscopic overview[J].Microsc Res Tech,2012,75(10):1437-1444.
[16]王丽,张会峰,赵志刚,等.大鼠骨髓内皮祖细胞的分离、培养与鉴定[J].中国组织工程研究,2012,16(10):1733-1736.
[17]Li M.Bone marrow-derived endothelial progenitor cells:Isolation and characterization for myocardial repair[J].Methods Mol Biol,2010,660:9-27.
[18]何灿粲,计晓娟,余更生,等.过表达CXCR4的内皮祖细胞影响血管平滑肌细胞迁移、增殖的实验研究[J].第三军医大学学报,2017,39(15):1562-1568.
[19]王红娟,王娟,李楠,等.大鼠骨髓和外周血早晚期内皮祖细胞的分离培养和鉴定[J].中国组织工程研究,2013,17(6):1056-1063.
[20]Cheng CC,Chang SJ,Chueh YN,et al.Distinct angiogenesis roles and surface markers of early and late endothelial progenitor cells revealed by functional group analyses[J].BMC Genomics,2013,14:182.
[21]Cheng SM,Chang SJ,Tsai TN,et al.Differential expression of distinct surface markers in early endothelial progenitor cells and monocyte-derived macrophages[J].Gene Expr,2013,16(1):15-24.
[22]Kalka C,Masuda H,Takahashi T,et al.Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization[J].Proc Natl Acad Sci U SA,2000,97(7):3422-3427.
[23]Vasa M,Fichtlscherer S,Aicher A,et al.Number and migratory activity of circulating endothelial progenitor cells inversely corelate with risk factors for coronary artery disease[J].Circ Res,2001,89(1):E1-7.