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目的:研究小型猪外周血内皮祖细胞(EPC)的体外分离和定向分化、扩增培养方法,为EPC移植应用于临床提供实验依据。方法:密度梯度离心法从小型猪200mL新鲜全血中分离单个核细胞,用含血管内皮生长因子等各种添加剂的内皮细胞系列专用培养液,分别在包被与不包被的培养皿中进行贴壁培养,诱导其向内皮细胞分化,观察经过不同时间培养后的细胞生长情况并进行诱导分化后的生物学鉴定,包括细胞表型鉴定、DiI标记的乙酰化低密度脂蛋白(DiI-acLDL)摄取试验、超微结构鉴定、体外血管生成实验。结果:包被了贴壁因子的培养皿细胞贴壁及增殖均多于未包被组。前者第3-4d可观察到梭形贴壁细胞,10d后出现多个细胞簇,14d左右可观察到条索状、网状血管样结构,原代细胞培养21d左右接近融合并且呈典型的鹅卵石样排列。第7-14d有大于98%的细胞Flk-1、vWF、CD31表达阳性,CD34+细胞为(26.01±2.82)%,有大于95%的细胞DiI-acLDL摄取试验阳性,透射电镜可见特征性的Weible-Palade小体存在,在血管生成实验中,血管内皮生长因子显著促进EPC形成小管的数量与复杂程度,呈一定的量效关系。结论:密度梯度离心法结合贴壁筛选培养法可以用于体外分离外周血中EPC进行实验研究,EPC在一定的诱导培养条件下能分化成为血管内皮细胞,贴壁因子、血管内皮生长因子等对于体外培养EPC有很重要的作用。
OBJECTIVE: To study the isolation, differentiation and expansion of endothelial progenitor cells (EPCs) from porcine peripheral blood in vitro and to provide an experimental basis for clinical application of EPC transplantation. Methods: Mononuclear cells were isolated from 200 mL fresh whole blood of miniature pigs by density gradient centrifugation. The endothelial cells were cultured in coated and uncoated dishes respectively with various additives such as vascular endothelial growth factor The adherent cells were cultured in vitro and induced to differentiate into endothelial cells. The growth of cells cultured at different times was observed and the biological characteristics of the cells were induced and differentiated, including cell phenotype, DiI-labeled low density lipoprotein (DiI-acLDL) ) Uptake test, ultrastructure identification, in vitro angiogenesis experiment. Results: Petri dish coated with adherent factor had more adherent cells and proliferation than those without coating. Fusiform adherent cells were observed on the first 3-4 days, and many cells clustered after 10 days. The cord-like and reticular vascular-like structures were observed on the 14th day. The primary cells cultured for about 21 days were close to fusion and showed typical pebbles Arranged like. The number of Flk-1 positive cells was higher than 98% on the 7-14th day, the expression of vWF and CD31 was positive, the percentage of CD34 + cells was (26.01 ± 2.82)%, and the positive rate of DiI-acLDL was higher than 95% -Palade bodies exist, in the angiogenesis experiment, vascular endothelial growth factor significantly promote the number and complexity of EPC formation of tubules, showing a certain dose-effect relationship. Conclusion: The method of density gradient centrifugation combined with adherent screening culture can be used to isolate EPC from peripheral blood in vitro. EPC can differentiate into vascular endothelial cells, adherent factors and vascular endothelial growth factor under certain culture conditions In vitro EPC has a very important role.