肝纤维化大鼠骨髓源性内皮祖细胞的培养及鉴定

Culture and identification of bone marrow-derived endothelial progenitorcells in rats with liver fibrosis

目的建立肝纤维化大鼠骨髓源性内皮祖细胞(EPCs)的体外培养体系,为自体细胞移植促进肝血管再生、治疗肝纤维化提供合适的供体细胞。方法 Wistar大鼠皮下注射CCl4,制作肝纤维化模型;通过密度梯度离心法从肝纤维化大鼠骨髓中分离单个核细胞,并用差速贴壁培养法进行体外诱导培养成EPCs;流式细胞仪检测其表型和纯度,乙酰化低密度脂蛋白和结合荆豆凝集素双荧光染色鉴定其吞噬功能,血管网形成实验鉴定其成血管功能。结果细胞培养14 d后呈现铺路石样结构;EPCs(63.9%±2.15%,P<0.05)为CDl33/VEGFR2双阳性细胞;,EPCs具有成熟内皮细胞的吞噬功能,并能形成稳定的血管网样结构。结论利用本实验室培养体系,肝纤维化大鼠骨髓细胞能被成功诱导培养为具有特征性表型和功能的EPCs。

Objective To establish a culture-system of bone marrow-derived endothelial progenitor cells （EPCs） in liver-fibrosis rats as a suitable donor cell bank for the treatment of liver fibrosis and angiogenesis by auto-cellular transplant. Methods He mononuclear cells from bone marrow in liver-fibrosis rat were collected by density gradient separation. Then these cells were cultured and induced into EPCs in vitro differential adhesion. The phenotype and purity of EPCs were detected by flow cytometry. The phagocytosis function of EPCs was identified by stainning with DiI-ac-LDL and FITC-UEA-1. The ability of incorporation into the vascular network of EPCs was tested in Matrigel. Results The cobblestone structure of EPCs was observed after 14-days culture. About 63.9% ± 2. 15% （P 〈 0. 05） cells were CD133 ＋/VEGFR2 ＋ EPCs . These cells had phagocytosis function and formed primitive vascular tube-like structures when planted in matrigel. Conclusions By this culture-system, bone marrow cells in rats with liver fibrosis can be successfully cultured and induced into EPCs with characteristic phenotypes and functions.