淫羊藿素抑制人肝细胞癌BEL-7402细胞株新生血管的机制研究

Research on Inhibitation Mechanism of Icaritin on Human Hepatocellular Carcinoma Cell Line BEL-7402 Angiogenesis

目的:探讨淫羊藿素(ICT)抑制人肝细胞癌BEL-7402细胞新生血管的可能机制。方法:对人肝细胞癌BEL-7402细胞进行体外培养,经不同浓度淫羊藿素处理后,采用MTT法检测其对人肝细胞癌BEL-7402细胞增殖的影响;采用酶联免疫法(ELISA)检测色素上皮源性因子(PEDF)和血管内皮生长因子(VEGF)在细胞上清液中的含量;采用反转录酶-聚合酶链锁反应法(RTPCR)检测细胞中PEDF和VEGFmRNA的表达;采用Western-Blot法检测细胞中PEDF和VEGF的蛋白表达。结果:不同浓度淫羊藿素(10-6mol/L、10-7mol/L、10-8mol/L)作用24 h、48 h后,未见其对人肝细胞癌BEL-7402细胞增殖有明显作用。但是,淫羊藿素作用24 h后,BEL-7402细胞上清液中VEGF的含量明显降低(P<0.05或P<0.01),同时PEDF的含量明显升高(P<0.05或P<0.01),均呈一定剂量依赖性。RT-PCR检测显示,ICT能有效降低BEL-7402细胞中VEGFmRNA水平,同时升高PEDFmRNA的转录水平(P<0.05或P<0.01)。Western-Blot检测也显示,ICT能有效降低BEL-7402细胞中VEGF蛋白含量,同时升高PEDF蛋白含量(P<0.05或P<0.01)。结论:体外实验结果表明淫羊藿素抑制人肝细胞癌BEL-7402细胞株新生血管生成的作用可能与其上调PEDF的表达、同时下调VEGF的表达有关。

Objective： To investigate the associate inhibitation mechanism of Icaritin （ICT） on human hepatocellular carcinoma cell line BEL-7402 angiogenesis. Methods： BEL-7402 cells were cultured in vitro. With different concentrations of ICT, the effects on its proliferation was observed by MTr assay; The content of vascular endothelial growth factor （VEGF） and pigment epithelium-derived factor （PEDF） in the cultural supernatants was detected by Enzyme Linked Immunosorbent Assay （ELISA）; The content of VEGF mRNA and PEDF mRNA was detected by RT-PCR; The protein level of VEGF and PEDF was detected by Western-blot. Results： ICT showed no significant effect on the proliferation of BEL-7402 ceils after 24 or 48 h treatment. With different concentrations （10-6, 10-7 and 10-8 mol/L） of ICT treatment for 24 h, BEL-7402 cells had a significant lower ability to secret VEGF, but for PEDF, it is on the opposite situation （P〈0.05 or P〈0.01）. The results of RT- PCR showed that ICT could decrease the mRNA content of VEGF while evaluate mRNA of PEDF. The results of Western blot revealed that ICT reduced the protein content of VEGF and elevated PEDF. Conclusion： These findings indicate that ICT can inhibit the angiogenesis of BEL-7402 cells in vitro, which may be related to the increased activity and synthesis of PEDF in BEL-7402 ceils, and also associated with the decreased activity and synthesis of VEGF.