猪外周血内皮祖细胞的分离培养及生物学特性

Isolation and culture of porcine endothelial progenitor cells from peripheral blood and their biological characteristics

目的从小型猪外周血中分离、培养内皮祖细胞(EPCs),并进行相关鉴定,分析其生物学特性。方法采用密度梯度离心法获得小型猪外周血单个核细胞并进行体外传代培养,分别采用倒置相差显微镜观察、免疫荧光染色、流式细胞仪分析及Matrigel实验和DiI-acLDL摄取实验,对其细胞表型及功能进行相关鉴定。结果外周血单个核细胞在培养过程中出现梭形贴壁和铺路石样形态;培养第14天,约80%的细胞DiI-acLDL吞噬实验阳性,且CD133、CD34、flk-1、CD31、CD144的表达阳性率分别达到25.1%、55.9%、97.7%、82.0%和95.4%;细胞培养于凝固的Matrigel表面7 d后,形成特征性的网格状结构。结论该实验成功从小型猪外周血单个核细胞中分离培养出EPCs,在其体外扩增诱导的过程中,表达EPCs和成熟内皮细胞的特征性标志。

Objective To isolate and culture endothelial progenitor cells （EPCs） from porcine peripheral blood, and to identify and evaluate their biological characteristics in vitro. Methods Peripheral blood mononuclear cells （PBMCs） were isolated by ficoll density gradient centrifugation method from porcine peripheral blood, and were cultured and then identified by immunofluoreseent staining （DiI-acLDL）. Endothelial cell-specific markers （CD133, CD34, flk-1, CD31 and CD144） were determined with flow eytometry analysis. Matrigel experiment was used to observe the function of EPCs. Results During the period of culture, cells became spindle-shaped and displayed cobble-stone morphology. The positive cells of DiI- aeLDL accounted for about 80% of the attached cells at d 14. The positive expression rates of CD133, CD34, flk-1, CD31 and CD144 were 25.1% , 55.9% , 97.7% , 82.0% and 95.4% , respectively. Matrigel experiment showed distinctive gratieule shape after 7 d. Conclusion EPCs can be isolated and cultured from PBMCs in porcine peripheral blood. During the culture, EPCs and mature endothelial cell markers are expressed.