血管内皮生长因子在肝癌血管内皮细胞体外管腔形成中量效和时效局限性的实验研究

摘要

Objective Based on tube formation model in vitro,to investigate the effects of vascular endothelial growth factor(VEGF)with dose- and time- dependent on the tube formation of tumor-derived endothelial cells (TECs),and to analyze the potential mechanism by which poor outcome of VEGF-target anticancer drug in the clinical application. Methods Isolation of tumor endothelial cells(TECs,CD31 positive cells)were treated by u-sing CD31- magnetic beadsfrom surgery tumor tissues of the liver cancer patients. Isolated TECs were co-cultured with different concentrations of VEGF165 on the matrigel in the 96-well plates. The tube formation with different concentration of VEGF at several time points was evaluated,respectively. Human umbilical vein endothelial cells (HUVECs)were served as the normal control. Results 1)TECs appeared spindle shape under the culture among which 96% of the cells were CD31 positive、CD31 a pan marker of endothelial cells;2)TECs nearly did not occur tube formation under co-culture with the basic condition containing 2 ng·mL - 1 VEGF165,while HUVECs showed the capillary tubule-1ike formation as well as a lot of branches. When 10 ng·mL - 1 VEGF165 was added in the culture,at 4 h TECs alike HUVECs appeared the tube formation that was at 6 h increased by 6 folds comparing to the basic condition culture(P ﹤ 0. 001). Interestingly,when 20 ng·mL - 1 VEGF165 were added in the culture,at 6 h,TECs occurred a little tube formation that was decreased by 4. 5 folds comparing to the co-culture with 10 ng·mL - 1 VEGF165(P ﹤ 0. 001). 3)When TECs were co-culture with 10 ng·mL - 1 VEGF165,tube formation showed at 4 h,6 h,8 h at which time point the tube formation in fact appeared reduction,and even disappeared at 20 h,while the clear tubes and branches were still existing in HUVECs control. Conclusion Our data demonstrate that the dose- and time- dependent effectiveness of VEGF on the tube formation of TECs from human liver cancer is limited. The higher concentration of VEGF may inhibit tube formation in vitro of TECs but not HUVECs. The time length of the tube formation is much shorter by TECs than by HUVECs. We proposal a potential mechanism by which poor outcome of VEGF-target anticancer drug in the clinical application may related to VEGF features as men-tioned above.%目的：利用体外管腔形成模型，研究血管内皮生长因子（VEGF）对肿瘤血管内皮细胞（TECs）调控的精准性，探讨以 VEGF 为靶点的抗肿瘤药物在临床应用中出现缺陷的潜在机制。方法采用 CD31免疫磁珠分选人肝癌标本中的 TECs，在含 Matrigel 基质胶的96孔板中，分别与不同浓度 VEGF 共培养，观察不同时间的 TECs 体外管腔形成能力，以正常人脐静脉血管内皮细胞（ HUVECs）为对照。结果1）体外分离培养的 TECs 拟血管内皮细胞梭状形态，其96％的细胞表达内皮细胞特异性标记 CD31；2）TECs 在微小量（基础培养条件，2 ng·mL -1）VEGF165时，体外管腔形成较少或不成腔，而对照组 HUVECs 却能形成明显的管腔并分支成网；当附加10 ng·mL -1 VEGF165时，在4 h 观察到 TECs 如同 HUVECs 形成了管腔，在6 h 出现明显的分支网；然而，当附加20 ng·mL -1 VEGF165时，在6 h 观察到 TECs 形成的管腔明显减少；统计分析显示，10 ng·mL -1 VEGF165组 TECs 成管能力比2 ng·mL -1 VEGF165组高出6倍（ P ﹤0.001），而20 ng·mL -1 VEGF165组 TECs 成管能力显著下降至10 ng·mL -1 VEGF165组的4.5倍（P ﹤0.001）；3）10 ng·mL -1 VEGF165组在培养4、6、8 h 均可见 TECs 管腔形成，但以6 h 为显著；在20 h 时，TECs 的管腔消失，而对照组 HUVECs 却还有明显的管腔分支网。结论肝癌 TECs 体外管腔形成对 VEGF165既有依赖性但又有量-效局限性，过高 VEGF 浓度能抑制性影响 TECs（而不是 HUVECs）管腔形成能力。在 VEGF 的刺激下，TECs 体外管腔形成的时效性短于 HUVECs。我们的实验结果提示，临床应用抗 VEGF 的抗肿瘤药物疗效不一可能与 VEGF 在 TECs 体外管腔形成的量效和时效的局限性有关。