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背景:内皮祖细胞因其分离与培养的方法各不相同,在实验中难以重复。目的:探讨大量获取骨髓源性内皮祖细胞分离与培养的方法。方法:通过密度梯度离心法从4周龄SD大鼠骨髓中分离单个核细胞,使用EGM-2 MV培养基进行诱导培养,采用形态学特征观察、摄取Dil-Ac-LDL与结合FITC-UEA-1实验、免疫荧光化学鉴定其表面抗原CD133与VEGFR2等方法对其进行鉴定,并通过管腔形成实验观察形成管腔的能力。结果与结论:①形态学观察:分离的骨髓单个核细胞经诱导培养后,在生长的早期(8d左右)、晚期(15d左右)其细胞形态有一定差异,早期以纺锤形、三角形、圆形细胞多见,晚期以圆形、短梭形细胞多见。②摄取Dil-Ac-LDL与结合FITC-UEA-1实验:显示8,21d的细胞均为阳性。③免疫荧光化学染色:8d的细胞表达CD133、VEGFR2。④管腔形成实验:在Matrigel基质上15h左右能够生成血管样结构。结果表明:利用密度梯度离心法分离大鼠骨髓单个核细胞后以EGM-2MV进行诱导培养,经过鉴定证明获得的细胞符合内皮祖细胞的特征。这种方法能够简单、快速、可靠、大量地获取内皮祖细胞。
Background: Endothelial progenitor cells vary in their methods of isolation and culture and are difficult to replicate in experiments. Objective: To explore a large number of bone marrow-derived endothelial progenitor cells isolated and cultured methods. Methods: Mononuclear cells were isolated from bone marrow of 4-week-old SD rats by density gradient centrifugation and cultured in EGM-2 MV medium. Morphological characteristics were observed and the uptake of Dil-Ac-LDL and FITC-UEA- 1 experiments, immunofluorescence chemical identification of its surface antigen CD133 and VEGFR2 and other methods to identify them, and through the lumen formation experiment to observe the formation of lumen ability. RESULTS AND CONCLUSION: ① Morphological observation: After the cultured bone marrow mononuclear cells were induced to culture, there were some differences in cell morphology between the early stage (about 8 days) and the later stage (about 15 days). The morphology of spindle cells was spindle, triangle, More common cells, the late round, short spindle cells more common. ② uptake of Dil-Ac-LDL and binding FITC-UEA-1 experiments: 8,21 d showed positive cells. ③ immunofluorescence staining: 8d cells express CD133, VEGFR2. ④ lumen formation experiment: about 15h on Matrigel matrix can generate vascular-like structure. The results showed that the rat bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured with EGM-2MV. After identification, the obtained cells were found to be in accordance with the characteristics of endothelial progenitor cells. This method allows for easy, rapid, reliable and extensive acquisition of endothelial progenitor cells.