树鼩胸主动脉血管内皮细胞的分离培养与鉴定

Culture and Identification of Thoracic Aortic Vascular Endothelial Cells Isolated from Tree Shrews

目的体外分离、培养树鼩(Tupaia belangeri)胸主动脉血管内皮细胞(vascular endothelial cells, VECs),并对培养细胞进行初步鉴定,为新型实验动物树鼩的应用研究提供体外模型。方法无菌取幼龄树鼩胸主动脉,采用组织块培养法和胰蛋白酶、胶原酶连续消化法分别进行树鼩胸主动脉血管内皮细胞分离,经完全培养液培养,倒置显微镜观察细胞形态,并用抗Ⅷ因子抗体进行荧光染色鉴定细胞。结果经组织块培养法分离培养的血管内皮细胞,3 d有细胞贴壁,7 d后细胞从组织块中大量长出,15 d汇合为单层;酶消化法所得细胞12 h即贴壁,3～7 d生长较快,12 d汇合成单层,镜下观察发现细胞呈典型的铺路石状;两种方法分离所得原代细胞经体外培养传代,6代内,细胞生长均良好;利用血管内皮细胞标志分子Ⅷ因子进行免疫荧光鉴定后确定为阳性。结论本研究采用简便的方法成功分离到了树鼩胸主动脉血管内皮细胞,为血管内皮细胞相关研究提供了实验模型。

Objective To study the isolation and culture of cultured vascular endothelial cells(VECs) from tree shrews(Tupaia belangeri), and to identify the cultured cells, and establish an in vitro model for the study of new tree shrews. Method The thoracic aorta was isolated from the tree shrews by tissue culture and trypsin digestion, culturing in complete culture medium, and cells morphology was observed by inverted microscopy, identified by Ⅷ factor Immunofluorescence assay. Result Tissue culture method was used to separate the cultured vascular endothelial cells, 3 d cells adherent, 7 d after the cells from the tissue block in a large number of long, cells were confluent to monolayer at 15 days. The cells were digested by enzyme digestion and adhered at 12 h, and grew at 3-7 days. The cells were treated with microscopic observation and showed that the cells were typical paving stone. The two cells were isolated and cultured in vitro. The cell growth was good and the positive expression was confirmed by Immunofluorescence assay using the vascular endothelial cell marker molecule Ⅷ. Conclusion VECs were successfully isolated from the tree shrews, and the method of isolation for tree shrews’ intestinal epithelial cells was established, which makes it available for VECs-related study.