蜂毒素体外抑制内皮细胞血管生成及其作用机制

Effects of melittin on angiogenesis in human endothelial cells in vitro and its relative mechanism

目的:探讨蜂毒素对人脐静脉来源内皮细胞(ECV304)血管生成的抑制作用及其机制.方法:体外培养ECV304细胞,分别测定蜂毒素对其增殖、迁移及形成管状结构的影响.采用酶联免疫吸附法检测血管内皮细胞生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)的含量.应用实时荧光定量PCR方法检测ECV304细胞VEGFmRNA和bF-GFmRNA的表达.结果:经蜂毒素作用后,ECV304细胞的增殖明显受到抑制,24,48,72h的IC50分别为5.11,4.68,4.40mg/L.蜂毒素低、中、高浓度组和沙利度胺(TLD)组ECV304细胞迁移数目(19.44±6.54),(11.17±2.85),(4.22±1.83)和(18.28±5.29)个,明显低于空白对照组(42.33±9.63)个(P<0.01).蜂毒素低、中、高浓度组及TLD组形成管状结构的面积分别为(5947.22±973.72),(1558.33±281.06),(705.85±318.01)和(2928.92±735.67)μm2/视野,均低于空白对照组(7828.94±1202.54)μm2/视野(P<0.01).经蜂毒素处理后,ECV304细胞分泌VEGF及bFGF的功能明显下降.荧光定量PCR证实,蜂毒素可降低ECV304细胞VEGFmRNA和bFGFmRN的表达.结论:体外实验结果表明,蜂毒素具有抑制ECV304细胞血管生成的作用,这种效应与血管内皮细胞VEGF和bFGF的活性降低及其合成受到抑制有关.

AIM： To investigate the effects of melittin on angio- genesis formed by human umbilical vein endothelial cells in vitro and the relative mechanisms. METHODS： Human umbilical vein endothelial cell line ECV304 was cultured in vitro. Using MTT assay, Transwell chamber assay, and Matrigel assay, the effects of melittin on the proliferation, migration and formation of capillary-like structure of ECV304 cells were evaluated. The con- centrations of vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF） in the supematant of ECV304 cells were examined by ELISA. Real time fluorescent quantative PCR was used to detect the mRNA expressions of VEGF and bFGF in ECV304 cells. RESULTS： Melittin inhibited the proliferation of ECV304 cells markedly, and ICs0 of 24, 48 and 72 h were 5.11, 4.68 and 4.40 mg/L, respectively. The number of migrated cells treated with melittin of the concentrations of 2. O, 4. O, 8.0 μg,/mL and thalidomide were 19.44 ± 6.54, 11.17 ± 2.85, 4.22 ± 1.83 and 18.28 ± 5.29, respectively, all significantly lower than that of the control group （42.33 ± 9.63, all P 〈 O. O1 ）. The area of capillary-like structure of the thalido- mide group was （2928.92 ± 735.67） μm^2/field, and those of the groups of melittin of the concentrations of 2.0, 4.0, and 8.0 μg/ mL were （5947. 22 ± 973. 72）, （1558. 33 ± 281. 06） and （705.85 ± 318.01 ） μm^2/field, respectively, all significantly less than that of the control group [ （7828.94 ± 1202.54） μm^2/ field, P 〈 0. O1 ]. ELISA assay showed that the secretion of VEGF and bFGF of ECV304 cells was significantly decreased after treatment with different concentrations of melittin and thalido-mide. Real time fluorescent PCR showed that the mRNA expres- sions of VEGF and bFGF in ECV3Od. cells of the melittin groups and thalidomide group were both significantly lower than those of the control group （ P 〈 O. O1 ）. CONCLUSION ： Melittin inhibits angiogenesis formed by ECV3Od. cells in vitro, which may be closely associated with the downregulation of the expressions of VEGF and bFGF in endothelial cells.