肿瘤源性血管内皮细胞的培养及其细胞生物学特性鉴定  

Culture and cell biologyical characteristics of tumor-derived endothelial cells

作　　者：蒋力[1] 何勇[1] 蒋耀光[1] 

机构地区：[1]第三军医大学大坪医院胸心外科中心,重庆400042

出　　处：《重庆医学》2009年第24期3102-3104,3195,共4页Chongqing medicine

基　　金：国家自然科学基金资助项目(No.30600611);第三军医大学青年科研基金资助项目(XG200540)。

摘　　要：目的探讨肿瘤来源血管内皮细胞的诱导和体外培养方法，并对其进行细胞生物学特性鉴定。方法胰蛋白酶灌流消化法收集人脐静脉内皮细胞（HUVEC），采用肿瘤细胞的培养上清液诱导HUVEC增殖，制备肿瘤衍生的血管内皮细胞（Td—EC）。采用形态学观察和荧光染色Ⅷ因子相关抗原，鉴定血管内皮细胞，用RTPCR检测肿瘤血管内皮细胞标志物（TEM）的表达，采用迁徙试验、透射电镜、流式细胞仪细胞周期分析等方法检测、比较HUVEC和Td—EC的生物学特性。结果原代培养的HuVEC约于24h完全贴壁，4～5d后融合成单层铺路石样结构。Ⅷ因子荧光染色证实培养的细胞是HUVEC。经肿瘤细胞上清液诱导后，用RT-PCR检测到TEM1和TEM8 mRNA在Td—EC中的表达；Td—EC细胞迁徙能力较HUVEC显著增强；Td—EC细胞增生活跃，G0～S期细胞比例明显增高，具有肿瘤血管内皮细胞特性。结论胰蛋白酶灌流消化法可获得大量连续传代扩增的HUVEC，并可诱导生成肿瘤源性的血管内皮细胞。Objective To research tumor-derived endothelial cells（Td EC） induced by the source of huraan umbilical vein endothelial cells （HUVEC） in vitro methods,and to identify its cell biological characteristics. Methods We cultured human umbilical vein endothelial cells （HUVEC） and differentiated them into Td-ECs after co-cultured with supernatants of A549 cells, and identifid HUVEC with observation and fluorescence staining Ⅷ-related antigen,examined its cell biological characteristics in vitro with the cell migration assay,transmission electron microscopy （TEM） and flow cytometry assay. Results The primary culture cells from human umbilical vein were observed completely wall sticked in 24h,4 5d later,they fused like some structure of monolayer flagstone,and the Ⅷ factor associated antigens＇ fluorescence staining confirmed that the cultured cells were endothelial cells. After the cells were induced by culture supernatants of A549 cells,we could detect the mRNA of TEM1 and TEM8 in the Td-Ec with RTPCR. Td-EC had more active cellular proliferation and ceil migration capacity, G0/G1 and S phase proportion of Td-Ee was higher than HUVEC,so,these cells had some characteristics of Td-Ec. Conclusion This study reveals a method to get substantial endothelial cells,which could induce Td-Ec.

关 键 词：人脐静脉内皮细胞 肿瘤内皮抗原 Ⅷ因子 细胞迁徙 

分 类 号：R73-3[医药卫生—肿瘤]