华蟾素注射液对人淋巴管内皮细胞增殖及管状结构形成的影响

The effect of cinobufacini injection on proliferation and tube-like structure formation of human lymphatic endothelial cells

背景与目的:华蟾素注射液是一种传统抗肿瘤中药制剂,目前药效机制尚不明确。本研究旨在探讨华蟾素注射液对人淋巴管内皮细胞(human lymphatic endothelial cells,HLEC)增殖、迁移和管状结构形成的影响。方法:通过绘制细胞生长曲线,观察华蟾素注射液对HLEC增殖的影响;通过细胞迁移实验观察华蟾素注射液对HLEC迁移的影响;通过基质胶实验观察华蟾素注射液对HLEC管状结构形成的影响;采用蛋白质印迹法(Western blot)观察华蟾素注射液对HLEC中VEGFR-3及HGF蛋白表达的影响。结果:随着华蟾素注射液药物浓度增加(0.105、0.21和0.42μg/mL),HLEC抑制率逐渐增高,迁移率分别为0.87±0.07、0.69±0.05、0.37±0.02,对照组为1±0.03;HLEC成管率为0.90±0.06、0.83±0.02、0.63±0.05,对照组为1±0.02,VEGFR-3/β-actin光密度比值为0.440±0.017、0.618±0.100、0.803±0.091,对照组为1.030±0.017;HGF/β-actin光密度比值为0.293±0.021、0.540±0.027、0.760±0.087,对照组为0.940±0.020(P<0.05)。结论:华蟾素注射液能够显著抑制HLEC增殖、迁移及管状结构形成,并下调VEGFR-3和HGF蛋白的表达,对VFGFR-3信号转导通路的影响可能是抑制肿瘤淋巴道转移的机制之一。

Background and purpose： The cinobufacini injection is a traditional antitumor drug. However, its mechanism is still unclear. The purpose of this study was to observe the effect of cinobufacini injection on proliferation, migration and tube-like structure formation of human lymphatic endothelial cells （HLEC）. Methods： Cell growth curve was used to observe the effect of cinobufacini injection on the proliferation of HLEC; Migration assay was used to observe the effect of cinobufacini injection on the migration of HLEC; Matrigel assay was used to observe the effect of cinobufacini injection on the tube-like structure formation of HLEC; Western blot was used to analyze the expression of VEGFR-3 and HGF in HLEC under the action of cinobufacini injection. Results： With the increase of cinobufacini injection concentration （0.105, 0.21 and 0.42 μg/mL）, HLEC inhibition rate gradually increased; Migration rates were 0.87±0.07, 0.69±0.05, and 0.37A0.02; and 1A0.03 in control group. HLEC into the tube rates were 0.90±0.06, 0.83±0.02, and 0.63±0.05; and 1±0.02 in control group. VEGFR-3/β-actin optical density ratios were 0.440±0.017, 0.618±0.100, and 0.803±0.091; and 1.030±0.017 in the control group. HGF/β-actin ratios of the optical density were 0.293±0.021, 0.540±0.027, and 0.760±0.087; and 0.940±0.020 in control group （P〈0.05）. Which showed that cinobufacini injection significantly inhibited HLEC proliferation （P〈0.05）, migration （P〈0.05） and tube- like structure formation in dose dependent ways（P〈0.05）. Cinobufacini injection significantly descreased the expression of VEGFR-3 and HGF in HLEC in dose dependent ways（P〈0.05）. Conclusion： Cinobufacini injection significantly inhibits HLEC proliferation, migration and tube-like structure formation, and the down-regulation of VEGFR-3 and HGF may contribute to the inhibition of cinobufacini injection on HLEC.