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脐血、外周血内皮祖细胞分化成内皮细胞的实验研究(PDF)

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2006年第1期
页码:
18-22
栏目:
基础研究
出版日期:
2006-01-01

文章信息/Info

Title:
Inducing cord blood and peripheral blood derived EPCs into endothelial cells in vitro
作者:
邵琴1; 王长谦1; 范华骅2; 姜萌1; 刘 嬿2; 聂晓绚2; 高跞2
1.上海交通大学附属仁济医院心内科，上海 200001；2.上海血液中心血液工程研究室，上海 200051
Author(s):
SHAO Qin1;  WANG Chang-qian1;  FAN Hua-hua2;  JIANG Meng1; LIU Yan2; NIE Xiao-xuan2; GAO Li2
1.Renji Hospital, Shanghai Jiaotong University, Shanghai 200001,China; 2. Blood Engineering Institute of Shanghai Blood Center, Shanghai 200051,China
关键词:
单个核细胞 ; 脐血; 血单个核细胞; 外周; 内皮祖细胞; 内皮细胞
Keywords:
umbilical cord blood mononuclear cells;  peripheral blood mononuclear cells;  endothelial progenitor cells;  endothelial cells
分类号:
R329.2; R543.3
DOI:
-
文献标识码:
A
摘要:
目的 探讨人的脐血、外周血内皮祖细胞(endothelial progenitor cells, EPCs)体外分离、纯化、诱导扩增和分化为内皮细胞的可行性，并检测其表型和功能。 方法 新鲜脐血和健康成年人的外周血，使用Ficoll密度梯度离心法得单个核细胞，在M199培养基中体外培养，3 d后去除悬浮细胞，继续培养，诱导EPCs增殖和分化。流式细胞仪检测EPCs标志CD34和内皮细胞特异性标志CD31表型，RT-PCR检测ecNOS, flk-1/KDR基因水平表达，免疫组化验证蛋白水平表达，并进一步通过NO活性的变化检测内皮细胞的功能。 结果 流式细胞仪检测，外周血单个核细胞(peripheral blood mononuclear cells, PBMC)刚分离时，CD34阳性表达率为（1.1±0.8）％，培养3 d后为（16.9±6.2）％。细胞形态观察发现，刚分离的单个核细胞呈圆形，形态小，3 d后有明显集落形成，7 d后梭形细胞线样排列，随培养时间增加，细胞形态逐渐变大，呈现出典型铺路石样改变。脐血单个核细胞(umbilical cord blood mononuclear cells,CBMC)和 PBMC培养10 d后，CD31阳性表达率分别为（76±17）％和（82±9）％。RT-PCR检测有内皮细胞特异性成分ecNOS，flk1/KDR的表达。免疫组化染色，细胞膜和细胞浆中有弥漫性棕色出现，呈阳性反应，证实了蛋白水平的表达。培养10 d的贴壁细胞随着VEGF浓度增加，NO生成增加，具有内皮细胞的功能。 结论 脐血，外周血EPCs体外分离，纯化，诱导培养后的贴壁细胞表型检测，大部分细胞具有内皮系标志物，并具有产生NO功能。
Abstract:
AIM To investigate the feasibility of inducing human umbilical cord blood and peripheral blood derived EPCs into endothelial cells in vitro, and to test their phenotype and function. METHODS Mononuclear cells were isolated from human umbilical cord blood and peripheral blood by density gradient centrifugation, and cultured in M199 medium. After 3 days, nonadherent cells were removed, the culture was maintained. The EPCs specific surface mark CD34 and endothelial specific surface marker CD31 were assessed by fluorescence activated cell sorter (FACS) analysis. Endothelial specific ecNOS and flk-1/KDR were detected by immunocytochemistry staining, reverse transcriptionpolymerase chain reaction (RT-PCR), respectively. The endothelial function of the cells was determined by measuring nitric oxide (NO) production in response to different concentrations of VEGF. RESULTS CD34 positive cells were only （1.1±0.8）% when freshly isolated PBMC, and increased to （16.9±6.2）% after 3 days. When CBMC, PBMC were cultured in medium, a number of cell clusters appeared at 3 d, and spindleshaped cells were observed and linear cordlike structures were formed at 7 d. Gradually, the attached (AT) cells exhibited cobblestone morphology, which was characteristic of endothelial cells. At 10 d, CD31 positive cells were （76±17）％and （82± 9）％in cultured CBMC and PBMC, respectively. The expression of EC marks of ecNOS and flk1/KDR was detected by RT-PCR and further confirmed by immunocytochemistry staining. NO production increased with incremental dose of VEGF. CONCLUSION When isolated and cultured in special medium, PBMC and CBMC show EC-like morphology, express the marks of EC lineage and have EC function.

参考文献/References

［1］Kalka C, Tehrani H, Laudenberg B, et al. VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease［J］. Ann Thorac Surg，2000，70(3):829-834.

［2］ Jozkowicz A, Dulak J, Nanobashvili J, et al. Vasculogenesis- a new strategy for induction of peripheral neovascularization［J］. Acta Chir Austriac，2002,34(2):114-117.

［3］ Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis［J］. Science， 1997， 275（5302）:964-967.

［4］ Murohara T, Ikeda H, Duan J,et al. Transplanted cord bloodderived endothelial precursor cells augment postnatal neovascularization［J］. Clin Invest，2000 ,105(11):1527-1536.

［5］ Kalka C, Masuda H, Takahashi T, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization［J］. Proc Natl Acad Sci USA，2000，97（7）:3422-3427.

［6］ Shintani S, Murohara T, Ikeda H, et al. Augmentation of postnatal neovascularization with autologous bone marrow transplantation［J］. Circulation，2001，103（6）:897-903.

［7］ Risau W, Sariola H, Zerwes HG, et al. Vasculogenesis and Angiogenesis in Embryonic Stem Cellderived Embryoid Bodies［J］. Development，1998，102:471-478.

［8］ Mutin M, Canavy I, Blann A, et al. Direct evidence of endothelial injury in acute myocardial infarction and unstable angina by demonstration of circulating endothelial cells［J］. Blood,1999, 93(9):2951-2958.

［9］ Kaushal S, Gilad EA, Krtsine JG,et al. Fuctional smalldiameter neovessels created using endothelial progenitor cells expanded ex vivo［J］. Nature Medicine，2001，7（9）:1035-1040.

备注/Memo

备注/Memo:
收稿日期：2005-05-04.基金项目：国家自然科学基金项目资助（No.30170365） 通讯作者：王长谦，主任医师，博士 Email：wcqian@hotmail.com 作者简介：邵琴，主治医师，硕士 Tel：（021）632609302324 Email：qwywy@163.com
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