纳米金与量子点标记的VEGF、VEGFR分子作用研究

发布时间：2018-02-28 01:22

本文关键词： 近场光学显微镜量子点纳米金血管内皮生长因子　出处：《暨南大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的利用纳米金能够阻止具有肝素结合位点的血管内皮生长因子(Vascular endothelial growth factor, VEGF) 165与其受体VEGFR2结合的特点,基于近场光学显微镜结合量子点技术研究纳米金阻断VEGF165与其受体VEGFR2结合的分子作用机制。方法1.MTT法检测纳米金是否抑制含有肝素结合位点的VEGF 165诱导的血管内皮细胞增殖作用,同时用没有肝素结合位点的VEGF121作为对照；2.原子力显微镜(atomic force microscope, AFM)观察纳米金、VEGF作用前后人脐静脉血管内皮细胞(Human umbilical vein endothelial cells, HUVECs)表面超微结构变化；3.利用量子点(quantum dots, QDs)对内皮细胞膜表面VEGFR2与VEGF165分子标记,在加入不同浓度纳米金前后,应用高分辨率的共聚焦显微镜和近场光学显微镜探测这些分子间的相互作用；4.无血清培养人脐静脉血管内皮细胞(HUVECs),加入VEGF165,再加入不同浓度纳米金,作用5 min,采用Western blot方法检测细胞内血管内皮生长因子受体-2下游信号磷酸化PLC-yl蛋白的水平。结果1.血管内皮细胞增殖抑制实验：纳米金明显抑制VEGF165诱导的血管内皮细胞增殖作用,而对缺乏肝素结合位点的VEGF121没有明显抑制作用；2.AFM检测到纳米金与VEGF165、VEGF121作用前后血管内皮细胞超微结构变化,VEGF165诱导的血管内皮细胞表面出现许多大小约数百纳米的孔道或颗粒,细胞膜边缘铺展延伸。加入VEGF165、纳米金后,细胞表面孔道或颗粒大为减少。VEGF121诱导的血管内皮细胞在纳米金加入前后的表面超微结构无明显变化；3.共聚焦显微镜和近场光学显微镜探测到纳米金阻断了VEGF165与其细胞膜表面受体VEGFR2的结合。4.VEGF165浓度不变(10μg/L),随着纳米金溶液浓度的增加(125、250、500nmol/L),纳米金抑制PLC-γ1磷酸化作用越来越明显,而对缺乏肝素结合位点的VEGF121没有明显抑制作用。结论1.纳米金抑制了VEGF165促血管内皮细胞增殖的作用：2.纳米金能够阻止具有肝素结合位点的VEGF165与其受体VEGFR2的结合,阻止其下游的细胞信号传导,从而抑制了VEGF165诱导的血管内皮细胞增殖：3.结合量子点标记技术,高分辨率的近场光学显微镜是研究生物单分子间相互作用的有力工具。
[Abstract]:Objective to investigate the ability of gold nanoparticles to prevent vascular endothelial growth factor (VEGF165) from binding to its receptor VEGFR2 with heparin binding site. The molecular mechanism of gold nanoparticles blocking the binding of VEGF165 to its receptor VEGFR2 was studied based on near-field optical microscopy and quantum dot technique. Methods 1. The proliferation of vascular endothelial cells induced by VEGF 165 containing heparin binding site was detected by MTT assay. Atomic force microscope2. Atomic force microscope (AFM) was used to observe the surface ultrastructural changes of human umbilical vein endothelial cells (HUVECs) before and after the treatment of VEGF121. 3. Quantum dots were used to measure the surface ultrastructure of human umbilical vein endothelial cells (HUVECs). QDsto the VEGFR2 and VEGF165 molecular markers on the surface of endothelial cell membrane. High resolution confocal microscope and near-field optical microscope were used to detect the interaction between these molecules before and after different concentrations of nanocrystalline gold were added. Human umbilical vein endothelial cells (HUVECs) were cultured without serum and VEGF165 was added, then different concentrations of nanocrystalline gold were added. Western blot assay was used to detect the phosphorylation of PLC-yl protein in the downstream signal of vascular endothelial growth factor receptor-2 (VEGF-2) at 5 min. Results 1. Inhibition of vascular endothelial cell proliferation: gold nanoparticles significantly inhibited the proliferation of vascular endothelial cells induced by VEGF165. 2. However, there was no obvious inhibitory effect on VEGF121 without heparin binding site. 2. The ultrastructural changes of vascular endothelial cells induced by VEGF165 were detected before and after the treatment of nano-gold with VEGF165VEGF121. The surface of vascular endothelial cells induced by VEGF165 showed a number of pore channels or particles about hundreds of nanometers in size. Extend the edge of cell membrane. Add VEGF165. after gold nanocrystalline, The surface ultrastructure of vascular endothelial cells induced by VEGF121 had no obvious change before and after the addition of nano-gold. Confocal microscope and near-field optical microscope detected that nano-gold blocked VEGF165 and its effect. Binding of membrane surface receptor VEGFR2. 4. The concentration of VEGF165 remains unchanged at 10 渭 g / L ~ (-1). With the increase of the concentration of gold nanoparticles, the inhibition of PLC- 纬 _ 1 phosphorylation by nano-gold becomes more and more obvious with the increase of the concentration of gold nanoparticles at the concentration of 125 ~ 250 ~ 500nmol / L ~ (-1). However, there was no obvious inhibitory effect on VEGF121 without heparin binding site. Conclusion 1. Nanocrystalline gold inhibits the proliferation of vascular endothelial cells induced by VEGF165. Nanocrystalline gold inhibits the binding of VEGF165 with heparin binding site to its receptor VEGFR2 and inhibits its downstream cell signal transduction. 2. Therefore, the proliferation of vascular endothelial cells induced by VEGF165 was inhibited. Combined with quantum dot labeling technique, high resolution near-field optical microscope is a powerful tool for studying the interaction between biological monolayers.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

【参考文献】