大鼠内皮祖细胞的分离培养与鉴定

Isolation,cultivation and identification of endothelial progenitor cells from rat bone marrow

目的 研究大鼠骨髓来源的内皮祖细胞的分离培养和鉴定方法.方法 纤维连接蛋白提前包被培养瓶和培养板中的盖玻片.密度梯度离心法分离SD大鼠骨髓单个核细胞,EGM-2完全培养基,贴壁法,37℃, 5%CO2,饱和湿度的恒温培养箱中原代培养.部分培养瓶细胞消化后计数.其余培养瓶和培养板中盖玻片细胞继续培养,细胞近融合时传代,分别在第6、9、12和24天进行细胞鉴定.内皮祖细胞鉴定：免疫细胞化学的vWF和VEGFR-2染色;内吞DiI标记的乙酰低密度脂蛋白（DiI-AcLDL）和结合FITC标记的荆豆凝集素-1（FITC-UEA-1）的激光共聚焦显微镜观察.结果 培养细胞的形态学改变：骨髓单个核细胞种植24h后部分细胞开始贴壁、变大,倒置显微镜下贴壁细胞透亮度增强,并逐渐伸出伪足样突起,呈小杆状或梭形.培养第3天贴壁细胞开始增殖,呈＂集落＂样生长,外周梭形细胞较多.第6天梭形细胞显著增多,培养至第12天左右,梭形细胞首尾相连,呈“毛细血管”样排列.培养第6天的细胞数量级为10^6～10^7/瓶.内皮祖细胞的鉴定：激光共聚焦显微镜观察显示细胞DiI-acLDL和FITC-UEA呈红绿免疫双荧光阳性.培养第6天细胞VEGFR-2和vWF免疫细胞化学染色呈阳性.结论 大鼠的骨髓单个核细胞可以培养为内皮祖细胞,且数量级达到10^6～10^7,可以满足动物实验研究的需要.

Objective To investigate the methods of isolation, cultivation and identification of endothelial progenitor cells （EPCs） from rat bone marrow, as well as their ability of differentiating into endothelial cells. Methods The mononuclear cells were isolated from SD rat bone marrow and were cultured in vitro via adhesion selection methods. The expression of Von Willebrand factor （vWF） and VEGFR-2 were assessed by immunocytochemistry after 6d,gd, 12d and 24d cultivation. And the adherent cells were stained with DiI complexed acetylated low-density lipoprotein （DiI-acLDL） and fluorescein Ulex Europaeus agglutinin-1 （FITC-UEA-1）, and then scanned by laser scanning confocal microscope（LSCM） to confirm EPCs lineage. Results The adherent cells ,stretched and exhibited the clone-like morphology after 3d cultivation and proliferated faster then. The cell number reached to 10^6 --10^7 per culture bottle at 6d. Immunocytoehemistry stain showed that the adherent cells were positive for vWF and VEGFR-2 from 6d after culture. The cells could take up DiI-aeLDL, and bind to FITC-UEA-1, showed double positive fluorescence under LSCM. Conclusion EPCs are enriched in rat bone marrow and may exhibited some of the characteristics of endothelial eells（ECs） after 9d inducing culture in vitro. The cell number can meet the need of research. .