水蛭素对肝细胞癌HepG2细胞抑制作用机制探讨

Inhibitory effects of hirudin on HepG2 human hepatocellular carcinoma cells and its mechanism of action

目的研究水蛭素对肝细胞癌(hepatocellular carcinoma,HCC,简称肝癌)HepG2细胞抑制作用及其抗肝癌的分子机制。方法将含不同浓度(1 U/m L、2 U/m L、4 U/m L和8 U/m L)水蛭素的培养液作用于肝癌HepG2细胞,采用MTT法检测水蛭素对肝癌HepG2细胞增殖的影响,流式细胞仪检测水蛭素对肝癌HepG2细胞凋亡的影响,Transwell法检测水蛭素对肝癌HepG2细胞迁移、侵袭的影响,荧光定量PCR检测血管内皮生长因子(vascular endothelial growth factor,VEGF)基因的m RNA表达水平,Western blot检测VEGF基因的蛋白表达水平。结果与空白对照组比较,肝癌HepG2细胞增殖抑制率随水蛭素浓度(1 U/m L、2 U/m L、4 U/m L和8 U/m L)增加及作用时间(24 h、48 h、72 h)延长而增加,呈剂量-时间依赖效应(P<0.05)。不同浓度(2 U/m L、4 U/m L和8 U/m L)水蛭素作用48 h后,肝癌HepG2细胞凋亡率分别为(28.37±1.16)%、(40.27±0.97)%、(76.17±1.5)%,细胞侵袭个数分别为(204±9)个、(163±6)个、(94±4)个,细胞迁移个数分别为(86±5)个、(54±7)个、(20±5)个,细胞凋亡率、侵袭及迁移个数随水蛭素浓度增加而增加,呈浓度依赖性(P<0.05)。VEGF m RNA、蛋白的表达量随水蛭素浓度增加而明显下调(P<0.05)。结论水蛭素能抑制肝癌HepG2细胞增殖、凋亡、迁移及侵袭,其机制可能是通过下调VEGF表达。

Objective To study whether and how hirudin inhibits growth of the HepG2 human hepatocellular carcinoma cell line. Methods The antiproliferative effects of different hirudin concentrations on HepG2 cells were assessed using the MTT assay. The apoptosis rate was assessed by immunofluorescence, and migration and invasion ability were measured in a transwell assay. Real-time quantitative PCR was used to measure levels of VEGF mRNA,while Western blot was used to examine levels of VEGF protein. Results The inhibition rate of HepG2 cells increased with increasing hirudin concentration and action time in a dose-dependent manner（P〈0.05）. The apoptotic rate of HepG2 cells increased with increasing hirudin concentration at 48 h,the rate in each concentration group were as follows：（28.37±1.16）%,2 U/mL;（40.27±0.97）%,4 U/mL;and （76.17±1.5）%,8 U/mL（P〈0.05）,while hirudin decreased both the numbers of migrating cells（204±9 to 2 U/mL,163±6 to 4 U/mL,and 94±4 to 8 U/mL,P〈0.05）and invasive cells （86±5 to 2 U/mL,54±7 to 4 U/mL and 20±5 to 8 U/mL,P〈0.05）,and it significantly reduced levels of VEGF mRNA and protein（P〈0.05）.Conclusion Hirudin inhibits proliferation, apoptosis,migration and invasion of HepG2,and it may do so by down-regulating VEGF expression.