摘要
目的分析人肝癌血管内皮细胞(T3A)在表型和功能上的特性。方法从人原发性肝癌组织分离、纯化和培养血管内皮细胞,并分析比较其与盱窦内皮细胞(LSEC)在形态、表型和功能上的差别。用电子显微镜,检查细胞表面窗孔样结构;用流式细胞仪和荧光定量PCR,分析细胞表型的差别;用噻唑盐(MTT)比色法,分析比较TNFα诱导的细胞毒作用;用酶谱法和体外血管生成模型,评价细胞的血管生成能力;用酶联免疫吸附试验(ELISA),比较凝血和纤溶凶子的释放。结果人T3A细胞表达经典的血管内皮细胞标志vWF和CD31,并摄取乙酰化低密度脂蛋白,细胞表面存在大量无隔膜的窗孔样结构。表型分析发现,与肝窦内皮细胞相比,T3A细胞表达TNF受体p75增加,而表达TNF受体p55降低;ICAM-1的表达明显下调,而αvβ3和αvβ5的表达明显上调,这些改变与荧光定量PCR分析的结果一致。功能学研究发现,T3A细胞对TNFα诱导的细胞毒作用明显耐受,血管生成能力明显增强并具有高表达的凝血和纤溶活性。结论人T3A细胞在表型和功能上都具有一定的特殊性。
Objective To analyze the phenotypic and functional characteristics of endothelial （T3A） cells derived from human hepatocellular cell carcinoma. Methods Endothelial cells were isolated from human hepatocellular carcinoma specimens. The identification of T3A cells was performed by checking yon Willebrand Factor （vWF）, CD31, CD34 and Dil-Ac-LDL uptake. The cell surface fenestrations, a specific morphological feature of tumor derived EC, were investigated by scanning and transmission electron microscopy. The phenotypie characteristics of T3 A cells were analyzed by fluorescence-activated cell sorter （FACS） and were further conformed by real-time PCR at transcription level. Furthermore, tumor necrosis factor alpha （ TNFα ） -induced cytotoxicity was evaluated by 3-（ 4,5-dimethythiazolyl ） - 2, -diphenyl-2Htetrazolium-bromide （MTT） assay; Matrix metalloproteinase secretion was detected by zymography; Angiogenic ability in vitro was analyzed by culturing T3A cells in three-dimensional Matrigel plug. Coagulant and fibrinolytic activities were detected by enzyme-linked immunosorbent assay （ELISA）. Results The isolated T3A cells exhibited classic ＂spindle-shape＂ morphology and monolayer growth and contact inhibition properties. Immunofluorescent staining showed that T3A cells expressed vWF, CD31, CD34, and uptake of Dil-Ac-LDL at a high level. The cell surface fenestrations were observed on T3A cells by scanning and transmission electron microscopy. By FACS and real-time PCR, T3A cells were found to express αvβ3, αvβ5 and TNF receptor p75 at high levels, and TNF receptor p55 and ICAM-1 at low levels, as compared with those in human liver sinusoidal endothelial cells （LSEC）. In response to TNFα, LSEC exhibited a dose-dependent cytotoxicity, while T3A cells were resistant. Gelatin zymography showed that MMP-2 activity was higher in T3A cells than that in LSEC. In a three-dimensional plug of Matrigel, T3A cells exhibited stronger angiogenic ability as compared with LSEC. In addition, T3A cells released more tissue factor （TF）, tissue-type plasminogen activator （t-PA）, plasminogen activator inhibitor （PAI-1） and urine plasminogen activator （u-PA） than LSEC in response to TNFα. Conclusion Tumor-derived endothelial cells are phenotypically and functionally different from those derived from normal liver tissue.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2007年第6期419-423,共5页
Chinese Journal of Oncology
基金
国家自然科学基金资助项目(30230150)
国家"973"基金资助项目(2002CB513100)
国家"863"基金资助项目(2001AA221251)
关键词
肝细胞癌
血管内皮细胞
血管生成
Endothelial cells
Hepatocellular carcinoma
Angiogenesis
