中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (29): 4722-4727.doi: 10.3969/j.issn.2095-4344.2017.29.022

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

CD31免疫磁珠分选人脂肪内皮祖细胞的培养及鉴定

汤文燕1，2，杨印祥1，汪兆艳1，栾 佐1，2   

  1. 1解放军海军总医院，北京市 100048；2南方医科大学第三临床医学院，广东省广州市 510515
  • 修回日期:2017-05-04 出版日期:2017-10-18 发布日期:2017-11-08
  • 通讯作者: 栾佐，硕士，主任医师，解放军海军总医院，北京市 100048；南方医科大学第三临床医学院，广东省广州市 510515
  • 作者简介:汤文燕，女，1989年生，广东省佛冈县人，汉族，南方医科大学在读硕士，主要从事小儿神经系统疾病及干细胞移植方面的研究。

Culture and identification of endothelial progenitor cells from human adipose tissue with CD31 immunomagnetic beads

Tang Wen-yan1, 2, Yang Yin-xiang1, Wang Zhao-yan1, Luan Zuo1, 2   

  1. 1Navy General Hospital of PLA, Beijing 100048, China; 2The Third Clinical College of Southern Medical University, Guangzhou 510515, Guangdong Province, China
  • Revised:2017-05-04 Online:2017-10-18 Published:2017-11-08
  • Contact: Luan Zuo, Master, Chief physician, Navy General Hospital of PLA, Beijing 100048, China; the Third Clinical College of Southern Medical University, Guangzhou 510515, Guangdong Province, China
  • About author:Tang Wen-yan, Studying for master’s degree, Navy General Hospital of PLA, Beijing 100048, China; the Third Clinical College of Southern Medical University, Guangzhou 510515, Guangdong Province, China

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摘要：

文章快速阅读：

文题释义：
内皮祖细胞：内皮祖细胞是血管内皮细胞的前体细胞，亦称为成血管细胞，在生理或病理因素刺激下，可从骨髓动员到外周血，并参与损伤血管的修复。1997年，Asahara等首次证明循环外周血中存在能分化为血管内皮细胞的前体细胞,并将其命名为血管内皮祖细胞。
内皮祖细胞的鉴定：综合通过细胞形态学、细胞增殖能力、细胞表型以及细胞功能多方面对内皮祖细胞进行鉴定。体外扩增培养的CD31+细胞呈典型的铺路石样形态，且保持一定的生长增殖能力；采用CD34、血管内皮生长因子受体2及CD133等几种细胞表面分子鉴定内皮祖细胞。CD34是分子量为105-120 ku的跨膜细胞表面糖蛋白，选择性表达于造血干细胞及某些激活的血管内皮细胞，其功能是作为内皮细胞及造血前体细胞相互作用的黏附分子。CD133(即AC133)是造血干细胞及祖细胞选择性表达的胆固醇结合糖蛋白，相对分子质量为120 000，它的确切功能目前尚且未知。血管内皮生长因子受体2型也称为KDR(人)或Flk-1(鼠)，主要表达在内皮细胞上，其在成骨细胞,胰管细胞,神经细胞和肺上皮细胞也有少量的表达。CD31+细胞还可摄取乙酰化低密度脂蛋白(Dil-ac-LDL)，结合荆豆凝集素(FITC-UEA-Ⅰ)，且其在Matrigel基质胶中可形成管腔样结构。

 

摘要
背景：内皮祖细胞是成熟内皮细胞的前体细胞，可迁移到缺血组织，分化为成熟内皮细胞，发挥血管新生作用。在各种缺血性疾病具有广阔的应用前景，但其生物学特性及鉴定方法仍存在争议。
目的：探讨从人脂肪组织中分离培养内皮祖细胞的方法，并进行鉴定，为移植治疗缺血性疾病提供足量的细胞来源。
方法：采用酶消化法从人脂肪组织中分离培养出脂肪基质血管细胞(stromal vascular cells，SVF)，再通过免疫磁珠分选出CD31+细胞，体外扩增培养得到内皮祖细胞，并通过细胞形态、细胞生长增殖能力，细胞表型及细胞的功能多方面来完成鉴定。
结果与结论：①免疫磁珠分选后的CD31+细胞，体外扩增培养呈典型的铺路石样形态，且其保持一定的生长增殖潜能；②第5代CD31+细胞流式分析结果显示：CD31、CD34、VEGRR2、CD146和CD133表达量分别为98.84%、97.21%、64.07%、98.42%和2.55%，但几乎不表达造血干细胞表面标志CD45(1.1%)；③免疫荧光染色结果表明：CD31+细胞可摄取乙酰化低密度脂蛋白(Dil-ac-LDL)并可结合荆豆凝集素 (FITC-UEA-Ⅰ)。并且，CD31+细胞在Matrigel基质胶中可形成管腔样结构，具有体外成血管能力；④结果提示，通过酶消化法结合免疫磁珠分选法从人脂肪组织中分离培养内皮祖细胞是可行的。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID:0000-0003-2703-9741(栾佐)

关键词: 干细胞, 培养, 脂肪组织, 免疫磁珠分选法, 脂肪基质血管细胞(SVF), 体外成血管能力

Abstract:

BACKGROUND: Endothelial progenitor cells are precursor cells of mature endothelial cells, which can migrate to ischemic tissues and differentiate into mature endothelial cells, and then play an important role in vascular remodeling. Endothelial progenitor cells have wide application prospects in various ischemic diseases, but the biological characteristics and identification methods are still controversial.
OBJECTIVE: To investigate the methods of isolation and culture of endothelial progenitor cells from the human adipose tissue and to identify their biological features, in order to provide a sufficient source of cells for ischemic diseases. 
METHODS: Stromal vascular fraction cells were isolated from the human adipose tissue by enzymatic digestion, CD31+ cells were selected using immunomagnetic beads, and then cultured in endothelial basal medium-2 supplemented with the EGM-2-MV-SingleQuots. Endothelial progenitor cells were identified through detection of morphology, cell markers and cell functions. 
RESULTS AND CONCLUSION: (1) CD31+ cells were selected by immunomagnetic beads and then cultured and amplified in vitro, which displayed typical cobblestone-like morphology, and they maintain their proliferative ability. (2) Flow cytometry results showed that the CD31+ cells expressed CD31 (98.84%), CD34 (97.21%), VEGRR2 (64.07%), CD146 (98.42%) and CD133 (2.55%), but hardly expressed CD45 (1.1%), a hematopoietic stem cell marker. (3) The CD31+ cells were also found to incept Dil-ac-LDL and exhibit lectin binding capability. Furthermore, a lumen-like structure was formed in Matrigel, which has the ability of angiogenesis in vitro. To conclude, these results suggest that it is feasible to isolate and culture endothelial progenitor cells from the human adipose tissue by enzymatic digestion combined with immunomagnetic bead sorting.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Adipose Tissue, Antigens, CD31, Phenotype, Tissue Engineering

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