CAJ | 학술논문

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)24 h,分别加入预先孵育过夜的纳米金(1000 nmol/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、1000 nmol/L)纳米金各0.5 ml,分别与碱性成纤维细胞生长因子(bFGF,10 mg/L)0.5 ml,4℃孵育24 h;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HUVEC 5孔,每孔加入VEGF165(10μg/L)100μl;再加入不同浓度纳米金(125、250、500 nmol/L),100μl,作用5 min,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500 nmol/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30 nm.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.
목적 관찰납미금능부억제혈관내피세포증식,이급작용적분자궤제.방법 재96공판내,무혈청배양인제정맥혈관내피세포(HUVEC)24 h,분별가입예선부육과야적납미금(1000 nmol/L)+혈관내피생장인자(VEGF)165(10μg/L)、VEGF165각100μl,새서람(MTT)비색법관찰납미금대HUVEC증식적영향.취3충농도(250、500、1000 nmol/L)납미금각0.5 ml,분별여감성성섬유세포생장인자(bFGF,10 mg/L)0.5 ml,4℃부육24 h;재가입과포화농도적간소-경지당응효,리심후용bFGF항체검측상청액화침정물중bFGF함량변화.무혈청배양HUVEC 5공,매공가입VEGF165(10μg/L)100μl;재가입불동농도납미금(125、250、500 nmol/L),100μl,작용5 min,용Western blot방법측정린산화PLC-γ1단백.용AFM(원자력현미경)표정납미금여VEGF165작용후립경대소.결과 납미금+VEGF165조여VEGF165조적증식배수분별위1.75화4.25,표명납미금억제HUVEC적증식(t=14.421,P<0.01).납미금능구여구유간소결합위점적bFGF결합.VEGF165농도불변(10μg/L),수착납미금용액농도적증가,종125、250도500 nmol/L,납미금억제PLC-γ1린산화월래월명현.AFM관찰도납미금여VEGF165작용후,립경보편대우30 nm.결론 납미금여구유간소결합위점적VEGF165결합,억제료VEGF165적신호전도,종이억제혈관내피세포증식.
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 VEGFI65 (10 μg/L,100 μl). The effects of nanogold on the growth of HUVECs were assessed by MTT assay. Nanogold (0.5ml) 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 an-tibody. 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 rain. The phosphoryla-tion 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 + VEGFI65 group and VEGF165 group was 1.75 and 4.25, respectively, which indicated nano-gold 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 nanogold more and more inhibited phosphorylation of PLC-γ1. The size of nanogold acting with VEGF165 probed with AFM was generally over 30 nm. Conclu-sion Combining to VEGF165 with heparin binding domain, nanogold can inhibit VEGF165-induced sig-naling. Therefore nanogold inhibits the proliferation of vascular endothelial cells.