巨噬细胞移动抑制因子抗体对HepG2细胞增殖的影响

目的 研究巨噬细胞移动抑制因子(MIF)抗体对HepG2细胞增殖的影响,并探讨其作用机制.方法 四甲基偶氮唑盐法检测MIF抗体(50、100、200、400 μg/L)对HepG2细胞增殖的抑制作用,流式细胞仪检测细胞周期的变化,免疫组织化学法检测Cyclin D1蛋白表达,Westem blot检测血管内皮生长因子(VEGF)蛋白表达,酶联免疫吸附法检测MIF抗体对HepG2细胞分泌IL-6水平的影响.结果 MIF抗体可以抑制HepG2细胞生长并具有剂量和时间依赖性.MIF抗体可使细胞周期停滞于G<,0>/G<,1>期,不同浓度(0、50、100,200、400μg/L)MIF抗体作用48 h后,G<,0>/G<,1>期细胞比例分别为61.34%±1.08%,65.08%±2.71%,71.19%±1.19%、78.39%±1.00%,83.92%±0.51%.不同浓度MIF抗体作用后Cyclin D1蛋白表达率分别为26.06%±0.47%、22.34%±0.75%、18.06%±1.16%、14.03%±0.59%,明显低于对照组(29.51%±1.28%).不同浓度MIF抗体作用后,VEGF蛋白表达分别为21.22%±0.68%、19.64%±0.54%、18.04%±0.42%、16.59%±0.66%,明显低于对照组(23.23%±0.51%).MIF抗体对HepG2细胞分泌IL-6有抑制作用,随着MIF抗体浓度的增加,其IL-6分泌量也相应降低,分别为(210.67±9.31)pg/ml、(181.67±10.05)pg/ml、(160.50±6.60)pg/ml,(143.67±10.56)pg/ml,(118.01±7.48)pg/ml.结论 MIF抗体可抑制HepG2细胞的增殖,其可能机制是下调Cyclin Dl蛋白的表达,从而阻止其由G<,0>/G<,1>期向S期分化;也可能与其抑制VEGF蛋白的表达,减少IL-6的分泌有关.

Inhibition effect of MIF antibody on the growth of hepatocellular carcinoma cell HepG2 in vitro

Objective To investigate the inhibitory effect ofmacrophage migration inhibitory factor (MIF) antibody on the proliferation of HepG-2 cells and its mechanism. Methods HepG-2 cells were stimulated by different concentrations of MIF antibody (50, 100, 200 and 400 μg/L). The cell survival rateswere evaluated by MTT assay. The cell cycles were assessed by flow cytometry (FCM) analysis. Cyclin Dl protein expression was examined by immunohistochemical methods. Vascular endothelial growth factor (VEGF) protein expression was examined by Western blot. ELISA was applied to detect the influence of MIF antibody on the production of IL-6 of HepG-2 cells. Results HepG-2 cells were inhibited by MIF antibody in a dose and time dependent manner. FCM analysis showed the cell cycles of HepG-2 cells were blocked at G<,0>/G<,1> phase. With concentrations of MIF antibody of 0, 50, 100, 200, 400 μg/L, the percentages of cells in G<,0>/G<,1>phase at 48h were 61.34% ±1.08%, 65.08%±2.71%, 71.19%±1.19%, 78.39%±1.00%, 83.92%± 0.51%. With concentrations of MIF antibody of 50, 100, 200, 400 μg/L, the expressions of cyclin Dl protein were 26.06%± 0.47%, 22.34%± 0.75%, 18.06%±1.16%, 14.03%± 0.59%, significantly lower than those of the control group (29.51%±1.28%). When HepG-2 cells were treated with different concentrations of M1F antibodies of 50, 100, 200, 400 μg/L the expressions of VEGF protein were 21.22%±0.68%,19.64%±0.54%, 18.04%±0.42%, 16.59%± 0.66%, significantly lower than those of the control group (23.23%±0.51%). With MIF antibody concentrations of 0, 50, 100, 200, 400 μg/L, the exudation amount of IL-6 were correspondingly lower [(210.67±9.31) pg/ml, (181.67±10.05) pg/ml, (160.50± 6.60) pg/ml, (143.67±10.56) pg/ml, (118.01±7.48) pg/m]. Conclusion The proliferation of HepG-2 cells was inhibited after treatment with MIF antibody. The inhibiting effect is caused by blocking cell cycle progression at G<,0>/G<,1> phase, decreasing cyclin D1 protein expression, decreasing VEGF protein expression and decreasing the exudation amount of IL-6.