纳米金抑制血管内皮细胞增殖的分子机制

Molecular mechanism of nanogold inhibiting the proliferation of vascular endothelial cells

目的观察纳米金能否抑制血管内皮细胞增殖，以及作用的分子机制。方法在96孔板内，无血清培养人脐静脉血管内皮细胞（HUVEC）24h，分别加入预先孵育过夜的纳米金（1000nmol/L）＋血管内皮生长因子（VEGF）165（10μg/L）、VEGF165各100μl,噻唑蓝（MTT）比色法观察纳米金对HUVEC增殖的影响。取3种浓度（250、500、1000nmol/L）纳米金各0．5m1，分别与碱性成纤维细胞生长因子（bFGF，10mg／L）0．5ml，4℃孵育24h；再加入过饱和浓度的肝素-琼脂糖凝胶，离心后用bFGF抗体检测上清液和沉淀物中bFGF含量变化。无血清培养HUVEC5孔，每孔加入VEGF165（10M／L）100μl；再加入不同浓度纳米金（125、250、500nmol/L），100μl，作用5min，用Western blot方法测定磷酸化PLC-γ1蛋白。用AFM（原子力显微镜）表征纳米金与VEGF165作用后粒径大小。结果纳米金＋VEGF165组与VEGF165组的增殖倍数分别为1．75和4．25，表明纳米金抑制HUVEC的增殖（t=14．421，P〈0．01）。纳米金能够与具有肝素结合位点的bFGF结合。VEGF165浓度不变（10μg/L），随着纳米金溶液浓度的增加，从125、250到500nmol/L，纳米金抑制PLC-γ1磷酸化越来越明显。AFM观察到纳米金与VEGF165作用后，粒径普遍大于30nm。结论纳米金与具有肝素结合位点的VEGF165结合，抑制了VEGF165的信号传导，从而抑制血管内皮细胞增殖。

Objective To investigate whether nanogold can inhibit the proliferation of vascular endothelial cells, and to find out the molecular mechanism of their interaction. Methods Human umbili- cal vascular endothelial cells （HUVECs） were seeded in 96-well plates, serum-starved for 24 h, and then treated with nanogold （1000 nmol/L,100 μl） ＋ VEGF165 （10 μg/L, 100 μl） ,or VEGF165 （10 μg/L, 100μl） . The effects of nanogold on the growth of HUVECs were assessed by MTT assay. Nanogold （0.5 ml） at three different concentrations （250,500,1000 nmol/L） were preincubated with bFGF （10 mg/L, 0.5 ml） overnight at 4℃. bFGF was then precipitated from this complex with a saturating concentration of heparin-sepharose, and bFGF in the supernatant fraction or precipitated fraction was detected by bFGF antibody. VEGF165 （10 μg/L, 100 μl） and nanogold at three different concentrations （125,250,500 nmoL/ L, 100 μl） were added to one of 5 wells of serum-starved HUVECs, and acted for 5 min. The phosphorylation of PLC-γ1 was detected with Western blot. Atomic force microscopy （AFM） was used to examine the sizes in nano-scale of nanogold acting with VEGF165. Results The proliferation multiple of HUVECs in nanogold ＋ VEGF165 group and VEGF165 group was 1.75 and 4.25,respectively, which indicated nanogold inhibited the proliferation of HUVECs （t = 14. 421 ,P 〈 0.01 ）. Nanogold could bind to bFGF with heparin binding domain. When concentration of VEGF165 was constantly 10 μg/L, an increase in nanogold concentration from 125 to 500 nmol/L, as a result nanoguld more and more inhibited phosphorylation of PLC-γ1. The size of nanogold acting with VEGF165 probed with AFM was generally over 30 nm. Conclusion Combining to VEGF165 with heparin binding domain, nanogold can inhibit VEGF165-induced signaling. Therefore nanogold inhibits the proliferation of vascular endothelial cells.