缺氧条件下胶质瘤U251细胞培养上清液对血管内皮细胞形成管样结构的影响

Effect of Human Glioma U251 Cells-conditioned Culture Fluid on Capillary-like Tube Formation by Vascular Endothelial Cells under Hypoxia

目的探讨缺氧条件下胶质瘤细胞培养上清液对血管生成的影响及其与尿激酶型纤溶酶原激活物(uPA)的关系。方法在常氧、缺氧条件下对人胶质瘤U251细胞进行培养,随后取其上清液作为内皮细胞ECV304的条件培养液,并分别加入uPA及其受体(uPAR)、组织型纤溶酶原激活物(tPA)的单克隆抗体、αvβ3和αvβ5整联蛋白特异性阻滞剂,观察内皮细胞管样结构形成变化;免疫细胞化学法检测ECV304中uPA、uPAR、NF-κB的表达。结果缺氧组ECV304形成管样结构数量较常氧组增加(P<0.05),且随上清液浓度增加差异更明显;uPA、uPAR单克隆抗体可抑制此促进作用,而tPA抗体、αvβ3和αvβ5则无此作用;缺氧培养U251的上清液能刺激ECV304中uPA、uPAR、NF-κB表达。结论在缺氧条件下培养胶质瘤细胞培养液的上清液对血管内皮细胞管样结构形成有促进作用,且与uPA、uPAR的表达有关,与tPA、αvβ3和αvβ5整联蛋白的关系不密切。

Objective To explore the effect of glioma cells conditioned culture fluid on angiogenesis by vascular endothelial cells cultured under hypoxia. Methods Human glioma U251 cells were cultured in the DMEM culture fluid containing 10% calf serum, from which the supernatant in different concentration served as glioma cells-conditioned culture fluid where vascular endothelial ECV304 cells were cultured under hypoxia and nonnoxia. The capillary-like tube formations by ECV304 cells in the culture fluid without or with urokinase plasminogen activator （uPA）, uPA receptor （uPAR） and tissue plasminegen activator （tPA） monoclonal antibody and the inhibitor of intcgrin, αvβ3 and αvβ5 were observed. The expression of uPA, uPAR and NF-kB were determined by immunohistochemical technique. Results The number of the capillary-like tubes in hypoxia group was significantly more than that in normoxia group （P〈0.05）, and their number was dependent on the concentration of the conditioned culture fluid. The tube formations were blocked by uPA or uPAR antibody, not tPA antibody and the inhibitor of αvβ3 and αvβ5. The upregulation of uPA, uPAR and NF-kB expressions in ECV304 cells were produced by U251-conditioned culture fluid. Conclusions The capillary-like tube formation by vascular endothelial cells may be promoted by glioma-conditioned culture fluid under hypoxia, and is related to the expression of uPA and uPAR in vascular endothelial cells.