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|本期目录/Table of Contents|

小鼠脾脏内皮祖细胞的分离和培养(PDF)

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2006年第5期
页码:
532-535
栏目:
基础研究
出版日期:
2006-10-25

文章信息/Info

Title:
Culture of mice endothelial progenitor cells derived from spleen
作者:
赵晓辉; 黄岚; 尹扬光; 周健; 方玉强; 朱光旭; 陈剑飞; 刘兰; 崔斌
第三军医大学新桥医院全军心血管内科中心,重庆 400037
Author(s):
ZHAO Xiao-hui;  HUANG Lan;  YIN Yang-guang; ZHOU Jian;  FANG Yu-qiang;  ZHU Guang-xu;  CHEN Jian-fei;  LIU Lan;  CUI Bin
Department of Cardiovascular Centre, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China
关键词:
内皮祖细胞; 细胞培养; 脾脏 ; 小鼠
Keywords:
endothelial progenitor cell; cell culture; spleen; mice
分类号:
R363
DOI:
-
文献标识码:
A
摘要:
目的 探讨小鼠脾脏内皮祖细胞(endothelial progenitor cell, EPC)的体外分离,定向分化和培养方法。方法 梯度密度离心法从小鼠脾脏中分离单个核细胞，用含血管内皮生长因子的M199培养液培养，每4 d更换培养基去除非贴壁细胞，观察经过不同时间培养后的细胞形态、结构和功能变化。结果 培养4 d可发现梭形贴壁细胞，7 d后细胞成集落状，14 d左右可观察到条索状、网状血管样结构，原代细胞培养21 d左右接近融合并呈典型的鹅卵石样排列。培养7 d透射电镜可见特征性的WeiblePalade小体存在，细胞CD31表达阳性，细胞DiILDL摄取试验阳性，FITCUEAI染色阳性；流式细胞仪检测细胞Sca1 及VEGFR2 的总阳性率分别为(34.6 ±3.8)%和（50.5±4.9）%，且该细胞具有体外成血管能力。结论 通过密度梯度离心联合贴壁筛选可以获得脾脏EPC。
Abstract:
AIM To culture endothelia1 progenitor cells (EPC) from mice spleen in vitro. METHODS spleen cell suspension was prepared by gradient centrifugation of Ficoll and mononuclear cells in the middle layer were collected and then put into M199 culture medium containing VEGF. The nonadhere cells were excluded every four days by changing the medium, and the morphology, structure and function of the cells were studied. RESULTS The spindle cells were found on the fourth day. On the seventh day, the cluster of cells appeared. The network structure similar with blood vessel were found 7 days later. The cells looked like "cobblestone" on the 21st day. Under transmission electron microscope, theseventhday cells displayed Weibe1Palade bodies which were the characteristic of endothelium. Immunological cells staining with CD31 was positive. EPC were characterized as adherent cells, double positive for DiLDL uptake and letin binding by direct fluorescent staining. Respectively, the cells were both positive for Sca1 and VEGFR2 identified by flow cytometry. CONCLUSION Density gradient centrifugation combined with adherence cells filtration is an effective way to obtain EPC derived from mice spleen.

参考文献/References

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

［2］ Vasa M，Fichtlseherer S，Aicher A，et al． Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease［J］．Circ Res，200l，89（1）：El-E7．

［3］ AsaharaT．Masuda H， Tak ahashi T，et al．Bone marrow origin of endothelial progenitor ceUs responsible for postnatal vasculogenesis in physiological and pathological neovascularization［J］．Circ Res，1999，85(3)：22l-228．

［4］ Hur J, Yoon CH，Kim HS，et al．Characterization of two types of endothelial progenitor cells and their different contributiom to neovasculogenesis［J］．Arterioscler Thromb Vasc Biol，2004，24(2)：288-293．

［5］ Iwakura A,Luedemann C,Shastry S, et al. Estrogenmediated, endothelial nitric oxide synthase–dependent mobilization of bone marrow–derived endothelial progenitor cells contributes to reendothelialization after arterial injury［J］． Circulation, 2003，108(25)：3115-3121.

［6］ 江德勤，田杰，白永虹，等. 骨髓间充质干细胞移植在心肌病大鼠体内存活、分化的实验研究［J］. 心脏杂志，2005,17(1) 4-7.

［7］ 王晓旭，于玲范，姜雪松，等. 骨髓干细胞对急性梗死心肌修复的实验研究［J］. 心脏杂志，2005,17(1).31-33,36.

备注/Memo

备注/Memo:
收稿日期：2005-10-24.基金项目：国家自然科学基金项目资助（No.30270568）通讯作者：黄岚，教授，主要从事冠心病及其防治研究 Tel:(023)68774601 Email:lanhuang@hotmail.com 作者简介：赵晓辉，主治医师，讲师 Tel：（023）68774601 Email：zxhwn2002@yahoo.com.cn
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