肿瘤坏死因子α及雷公藤内酯醇调节Raji细胞内VEGF表达及基质胶.doc

肿瘤坏死因子α及雷公藤内酯醇调节Raji细胞内VEGF表达及基质胶 　 作者：崔国惠， 陈卫华， 薛克营， 刘芳， 陈燕 【摘要】 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ECV304（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25 nmol/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(Plt;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(10 ng/ml)和 TNF-α (10 ng/ml)处理的Raji细胞上清在基质胶中作用ECV304细胞后可促进血管形成，而雷公藤内酯醇(25 nmol/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。 【关键词】 肿瘤坏死因子-α； 雷公藤内酯醇； VEGF； Raji细胞； ECV304细胞； 血管形成 　　Vascular endothelial growth factor (VEGF) promotes angiogenesis and is associated with the invasion and metastasis of malignant tumours［1］. VEGF promotes acute myelogenous leukemia (AML) cell growth and survival, and may contribute to drug resistance［2，3］. Previous studies showed that TNF-α increased VEGF mRNA expression［4］ . Triptolide, a traditional Chinese medicine, has been reported to be effective in the treatment of auto-immune diseases, and it can also induce anti-neoplastic activity on several human tumour cell lines. Triptolide can inhibit VEGF expression［5］. Therefore, to better understand the possibilities of antiangiogenic tumour therapy, we investigate the effect of TNF-α and triptolide on the expression of VEGF in human B lymphoma Raji cells lines, and their effect on angiogenesis in human umbilical vein endothelial cells (HUVECs)-derived cell line (ECV304) to elucidate the mechanisms controlling the process of tumour neovascularization. 　　Materials and Methods 　　Drugs and reagents 　　Recombinant human TNF-α was purchased from Peprotechec, USA (#0604CY25,≥2×107 units/mg). Triptolide (molecular weight 360， from Tripterygium Wilfordii 98% (HPLC) solid )； Matrigel was purchase