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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 possible mechanism of icaritin (ICT) inhibiting neovascularization in human hepatocellular carcinoma BEL-7402 cells. Methods: Human hepatocellular carcinoma BEL-7402 cells were cultured in vitro and treated with different concentrations of icaritin. MTT assay was used to detect the effect on proliferation of human hepatocellular carcinoma BEL-7402 cells. ELISA was performed by enzyme-linked immunosorbent assay (ELISA) ) Were used to detect the contents of PEDF and VEGF in the cell supernatant. The expression of PEDF and VEGF mRNA in the cells was detected by reverse transcriptase-polymerase chain reaction (RTPCR) The protein expression of PEDF and VEGF in the cells was detected by Western-Blot. Results: The results showed that icaritin (10-6mol / L, 10-7mol / L, 10-8mol / L) for 24 h and 48 h showed no obvious effect on the proliferation of human hepatocellular carcinoma BEL-7402 cells effect. However, the content of VEGF in the supernatant of BEL-7402 cells was significantly decreased (P <0.05 or P <0.01) and the content of PEDF was significantly increased (P <0.05 or P <0.01) , Were dose-dependent. The results of RT-PCR showed that ICT could effectively decrease the level of VEGFmRNA in BEL-7402 cells and increase the transcriptional level of PEDFmRNA (P <0.05 or P <0.01). Western-Blot also showed that ICT can effectively reduce the VEGF protein content in BEL-7402 cells and increase the PEDF protein content (P <0.05 or P <0.01). Conclusion: The results of in vitro experiments showed that icaritin inhibited the angiogenesis of human hepatocellular carcinoma BEL-7402 cells by up-regulating the expression of PEDF and down-regulating the expression of VEGF.