大鼠脾脏与骨髓源内皮祖细胞生物学特性的对比研究

Biological characteristics of endothelial progenitor cells derived from rat spleen and bone marrow

目的体外分离培养Sprague Dawley(SD)大鼠脾脏和骨髓来源内皮祖细胞(EPCs),并比较其生物学特性。方法采用密度梯度离心法,从健康4周龄SD大鼠脾脏和骨髓中分离出单个核细胞,体外诱导分化为EPCs,对比分析两种EPCs的形态、增殖、迁移、黏附和一氧化氮(NO)分泌能力。结果从脾脏和骨髓均可分离获得大量单个核细胞,但骨髓组织可获得的细胞数量更多,两种来源的细胞在体外诱导培养均可形成类似于血岛样集落,绝大部分细胞吞噬DiI荧光标记的乙酰化低密度脂蛋白和绿色荧光标记的荆豆凝集素双染为阳性;骨髓来源的EPCs在细胞增殖、迁移、黏附和NO分泌能力等方面均显著高于脾脏来源的EPCs。结论骨髓较脾脏更适宜作为体外培养EPCs的组织来源。

Objective To compare the biological characteristics of endothelial progenitor cells derived from spleen and bone marrow in vitro. Methods Mononuclear cells derived from bone marrow and spleen of Sprague-Dawley（SD） rat were isolated by density gradient centrifugation,inoculated on fibronectin-coated culture flask and cultured with complete medium containing vascu- lar endothelial growth factor,basic fibroblast growth factor. Morphology was observed under an inverted microscope. FITC-labeled UIexeuropaeus agglutinin-I （FITC-UEA-I） and DiI-labeled acetylated low-density lipoprotein （DiI-ac-LDL） double staining of dif- ferentiating endothelial progenitor cells were identified by Laser confocal microscopy.Endothelial progenitor cells proliferation was measured by 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay, migration of endothelial progenitor cells by Transwell chamber assay, adhesion and nitric oxide（NO） secretion ability of endothelial progenitor cells by relevant experimental determination. Results The mononuclear cells derived from spleen and bone-marrow was isolated by density gradient separation and identified by differentiating into kinds of blood-island like cell clusters and staining FITC-UEA-1 and DiI-ac-LDL together ob- served by fluorescence microscope. On the part of the biological characteristics, such as proliferation, migration, adhesion and NO production,endothelial progenitor cells from the bone marrow is better than from the spleen. Conclusion Relative to the spleen, bone marrow is more suitable for the extraction of endothelial progenitor cells in vitro.