天冬多糖低氧下抑制肝癌作用的体外实验研究

Anticancer effects of deproteinized asparagus polysaccharide on hepatocellular carcinoma cells in hypoxia in vitro

目的探讨低氧环境下天冬多糖对人肝癌细胞生长的影响及初步作用机制。方法氯化钴(CoCl2)化学模拟低氧下培养4种人肝癌细胞株(HepG2、Hep3B、SMMC-7721及Bel-7402),加入不同浓度的天冬多糖(0mg/m L、0.9 mg/m L、1.2 mg/m L、1.8 mg/m L、2.4 mg/m L、2.7 mg/m L、3.6 mg/m L)培养,CCK-8法检测天冬多糖对4种人肝癌细胞株细胞增殖的影响,根据IC50选取0.9 mg/m L、1.8 mg/m L、2.7 mg/m L作为后续实验的天冬多糖低、中、高剂量浓度。后续实验4种细胞均设6组:常氧对照组只加入培养液培养;低氧对照组加入200μmol/L CoCl2培养;低氧低剂量组加入200μmol/L CoCl2+0.9 mg/m L天冬多糖培养;低氧中剂量组加入200μmol/L CoCl2+1.8 mg/m L天冬多糖培养;低氧高剂量组加入200μmol/L CoCl2+2.7 mg/m L天冬多糖培养;常氧高剂量组加入2.7 mg/m L天冬多糖培养。培养24 h后,Real-time PCR检测各组血管内皮生长因子(VEGF)、Bax、Bcl-2 mRNA表达情况,Western-blot方法检测各组缺氧诱导因子-1α(HIF-1α)、VEGF、基质金属蛋白酶-9(MMP-9)、Cleaved Caspase-3蛋白表达情况。结果低氧下天冬多糖可明显抑制人肝癌细胞株的生长,且抑制作用呈剂量依赖性。天冬多糖可明显抑制HIF-1α、VEGF、MMP-9蛋白表达及VEGF mRNA、Bcl-2mRNA表达,促进Cleaced Caspase-3蛋白及Bax mRNA表达,且低氧下高剂量作用最明显。结论天冬多糖在体外低氧条件下能抑制肝癌细胞的生长,其可能通过抑制HIF-1α/VEGF表达及肝癌细胞的侵袭、促进肝癌细胞凋亡起到抗肝癌的作用。

Objective It is to Study the inhibiting effect and mechanism of asparagus polysaccharide on human hepatocellular carcinoma cells in hypoxia environment. Methods Four kinds of human hepatocellular carcinoma cells lines( Hep G2,Hep3 B,SMMC-7721 and Bel-7402) were cultured in vitro in chemical hypoxia environment established by cobalt chloride,and they were cultured by asparagus polysaccharide of different concentrations( 0 mg/m L,0. 9 mg/m L,1. 2 mg/m L,1. 8 mg/m L,2. 4 mg/m L,2. 7 mg/m L,3. 6 mg/m L). The effect of aspartic polysaccharide on cell proliferation of four human hepatoma cell lines was detected by CCK-8 method. 0. 9 mg/m L,1. 8 mg/m L and 2. 7 mg/m L were selected according to IC50 as low,medium and high doses of aspartic polysaccharide in subsequent experiments. In the follow-up experiment,there were 6 groups of 4 kinds of cells: the normoxic control group was only added to the culture medium;the hypoxic control group was added with 200 μmmol/L Co Cl2;the low oxygen and low dose group was added with 200μmmol/L Co Cl2 + 0. 9 mg/m L aspartic polysaccharide culture. The low oxygen medium dose group was added with 200μmmol/L Co Cl2 + 1. 8 mg/m L aspartic polysaccharide culture;the low oxygen high dose group was added with 200μmmol/L Co Cl2 + 2. 7 mg/m L aspartic polysaccharide culture;the normoxia high dose group was added with 2. 7 mg/m L aspartate culture. After 24 hours of culture,Real-time PCR was used to detect the expression of vascular endothelial growth factor( VEGF),Bax and Bcl-2 mRNA in each group. Western-blot method was used to detect the expression of hypoxia-inducible factor-1α( HIF-1α),VEGF,matrix metalloproteinase-9( MMP-9),Cleaved Caspase-3 protein. Results Aspartame can significantly inhibit the growth of human hepatoma cell lines in a dose-dependent manner in hypoxia environment. Aspartame can significantly inhibit the expression of HIF-1α,VEGF,MMP-9 protein,VEGF mRNA and Bcl-2 mRNA,promote the expression of Cleaced Caspase-3 protein and Bax mRNA,and the highest dose was most obvious under hypoxia. Conclusion Aspartame can inhibit the growth of hepatoma cells under hypoxic conditions in vitro,which may play an anti-hepatocarcinal effect by inhibiting the expression of HIF-1α/VEGF and the invasion of hepatocarcinoma cells and promoting apoptosis of hepatoma cells.