肿瘤坏死因子α和雷公藤内酯醇调节Raji细胞内VEGF的表达及基质胶中ECV304细胞血管形成作用的研究

为了探讨肿瘤坏死因子（TNF—α）和雷公藤内酯醇作用Raji细胞VEGF（血管内皮细胞生长因子）的表达及VEGF与ECV血管形成的关系，采用MTT法检测雷公藤内酯醇抑制Raji细胞增殖的影响；用ELISA法对Raji细胞上清的VEGF定量；用基质胶上的内皮细胞网络形成检测ECV30d（人类脐静脉内皮细胞起源的细胞系）血管生成；用RT-PCR检测VEGF mRNA含量。结果表明：雷公藤内酯醇对Raji细胞增殖抑制作用呈时间和剂量依赖方式，最适合作用时间为24小时，24小时半数抑制浓度IC50为25nmol／L；Raji细胞上清VEGF的含量在TNF-α组明显高于空白对照组，雷公藤内酯醇处理组则明显低于空白对照组，两者比较有极显著差异（P〈0．01）；Raji细胞内VEGF mRNA主要为VEGF165和VEGF121，其含量在TNF—α处理组与空白对照组比较有增加，而雷公藤内酯醇抑制VEGF mRNA表达，并呈剂量依赖方式；Raji细胞上清、VEGF（10ng／ml）和TNF—α（10ng／ml）处理的Raji细胞上清在基质胶中作用ECV30d细胞后可促进血管形成，而雷公藤内酯醇（25nmol／L）处理的Raji细胞上清和1640培养基作为的对照组加入基质胶中ECV304细胞未见血管形成。结论：在TNF-α、雷公藤内酯醇作用Raji细胞过程中，TNF-α促进VEGF的表达而雷公藤内酯醇则抑制其表达；TNF-α处理的Raji细胞上清在基质胶中促进血管形成，而雷公藤内酯醇则抑制血管形成。

Effects of Triptolide and TNF-α on the Expression of VEGF in Raji Cells and on Angiogenesis in ECV304 Cells

In order to study the relation of antitumour mechanisms of triptolide with neovascularization, the effect of triptolide and tumour necrosis factor (TNF)-alpha on the expression of vascular endothelial growth factor (VEGF) in Raji cell lines and their effect on angiogenesis in human umbilical vein endothelial cells (HUVECs)-derived cell line ECV304 were investigated. The inhibitory rate of cells treated by triptolide detected by MTT; the ELISA was employed to study the VEGF content secreted by Raji cell lines; angiogenesis was tested by network formation of endothelial cells on Matrigel, and the mRNA level of VEGF was measured by RT-PCR. The results showed that treatment of Raji cells with triptolide resulted in significantly enhanced antiproliferative effects in dose- and time-dependent manner. The content of VEGF secreted by Raji cells was increased by TNF-alpha and was suppressed by triptolide (P < 0.01). The mRNA expressions of VEGF(165) and VEGF(121) (containing 165 and 121 amino acid residues, respectively) could be detected in all fractions. TNF-alpha augmented the expression of VEGF(165) and VEGF(121) mRNA when triptolide reduced the expression (P < 0.01). No network and cord were formed in control and triptolide group. There was tube formation on matrigel in the supernatants of Raji culture group and the supernatants groups treated by VEGF and TNF-alpha in Raji cell. It is concluded that the expressions of VEGF in Raji cells are increased by TNF-alpha and suppressed by triptolide. VEGF and TNF-alpha induce angiogenesis and triptolide inhibits angiogenesis in ECV304 cells.