地胆草提取物对肝癌血管生成拟态的作用研究

Effect of Elephantopus scaber L.extract on vasculogenic mimicry of hepatocellular carcinoma

目的研究地胆草提取物对肝癌HepG2细胞增殖、迁移、血管生成拟态的作用和机制.方法取对数生长期HepG2肝癌细胞,分为对照组(0.1%DMSO)和不同浓度实验组(地胆草提取物质量浓度分别为25,50,75,100,125,150μg·mL^(-1)).以噻唑蓝(MTT)比色法测定地胆草提取物对HepG2细胞增殖的影响;根据MTT结果,设对照组(0.1%DMSO),低浓度实验组(75μg·mL^(-1)),高浓度实验组(125μg·mL^(-1))开展后续实验.以细胞划痕实验和体外血管生成实验检测地胆草提取物对HepG2细胞迁移、血管生成拟态的影响;以蛋白质印迹(Western Blot)法和逆转录聚合酶链反应(RT-PCR)法检测血管内皮生长因子A(VEGFA)、基质金属蛋白酶-2(MMP-2)、MMP-3、MMP-9蛋白和mRNA的表达水平.结果地胆草提取物剂量依赖性抑制HepG2细胞的增殖,作用48 h的IC50为(89.47±1.54)μg·mL^(-1).对照组和低、高浓度实验组迁移率分别为(11.68±0.80)%,(5.53±0.44)%和(-8.17±1.95)%;体外成管实验显示,对照组、低、高实验浓度组管腔数量分别为39.80±5.97,32.60±2.61和11.00±5.83,管腔面积分别为1.24×10^(4)±2833.10,8257.70±2244.00和6674.50±1217.60,低、高浓度实验组与对照组相比,差异均有统计学意义(P<0.05或P<0.01).与对照组相比,低、高实验浓度组均可降低VEGFA、MMP-2、MMP-9的表达(均P<0.05),而对MMP-3的表达无明显影响.结论地胆草提取物可能通过下调VEGFA、MMP-2、MMP-9的表达发挥抑制肝癌细胞增殖、迁移及体外血管生成拟态的作用.

Objective To study the effects and mechanism of Elephantopus scaber L.on the proliferation,migration of HepG2 cells and vasculogenic mimicry in vitro.Methods HepG2 cells in logarithmic growth stage were divided into control group(0.1%DMSO)and experimental group with different doses(the doses of Elephantopus scaber L.extract were 25,50,75,100,125,150μg·mL^(-1)),MTT assay was used to determine the effect of Elephantopus scaber L.extract on the proliferation of HepG2 cells.According to the results of MTT,the follow-up experiments were divided into control group(0.1%DMSO),low dose experimental group(75μg·mL^(-1))and high dose experimental group(125μg·mL^(-1)).The effects of Elephantopus scaber L.extract on migration and angiogenic mimicry of HepG2 cells were detected by cell scratch test and in vitro angiogenesis assay.The protein and mRNA expression levels of vascular endothelial growth factor A(VEGFA),matrix metalloproteinase-2(MMP-2),MMP-3 and MMP-9 were detected by Western blot and reverse transcription polymerase chain reaction(RT-PCR).Results Elephantopus scaber L.inhibited HepG2 cells proliferation in a dose-dependent manner.After 48 hours of drug treatment,IC50was(89.47±1.54)μg·m L^(-1).The migration rates of control group and low,high dose experimental groups were(11.68±0.80)%,(5.53±0.44)%,(-8.17±1.95)%;the number of lumens in control group and low,high dose experimental groups were39.80±5.97,32.60±2.61,11.00±5.83,the lumen areas were 1.24×10^(4)±2833.10,8257.70±2244.00,6674.50±1217.60.Low,hight dose experimental groups compared with control group,the difference were all statistically significant(P<0.05,P<0.01).Compared with control group,the low and high dose experimental groups could reduce the expression of VEGFA,MMP-2 and MMP-9(all P<0.05),but had no significant effect on the expression of MMP-3.Conclusion The extract of Elephantopus scaber L.could realize the aim of inhibiting proliferation,migration and vasculogenic mimicry in vitro of hepatocellular carcinoma through down regulating the expression of VEGFA,MMP-2 and MMP-9.