發(fā)布時(shí)間：2018-02-28 01:22

  本文關(guān)鍵詞： 近場(chǎng)光學(xué)顯微鏡 量子點(diǎn) 納米金 血管內(nèi)皮生長(zhǎng)因子　出處：《暨南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的利用納米金能夠阻止具有肝素結(jié)合位點(diǎn)的血管內(nèi)皮生長(zhǎng)因子(Vascular endothelial growth factor, VEGF) 165與其受體VEGFR2結(jié)合的特點(diǎn),基于近場(chǎng)光學(xué)顯微鏡結(jié)合量子點(diǎn)技術(shù)研究納米金阻斷VEGF165與其受體VEGFR2結(jié)合的分子作用機(jī)制。 方法1.MTT法檢測(cè)納米金是否抑制含有肝素結(jié)合位點(diǎn)的VEGF 165誘導(dǎo)的血管內(nèi)皮細(xì)胞增殖作用,同時(shí)用沒(méi)有肝素結(jié)合位點(diǎn)的VEGF121作為對(duì)照；2.原子力顯微鏡(atomic force microscope, AFM)觀察納米金、VEGF作用前后人臍靜脈血管內(nèi)皮細(xì)胞(Human umbilical vein endothelial cells, HUVECs)表面超微結(jié)構(gòu)變化；3.利用量子點(diǎn)(quantum dots, QDs)對(duì)內(nèi)皮細(xì)胞膜表面VEGFR2與VEGF165分子標(biāo)記,在加入不同濃度納米金前后,應(yīng)用高分辨率的共聚焦顯微鏡和近場(chǎng)光學(xué)顯微鏡探測(cè)這些分子間的相互作用；4.無(wú)血清培養(yǎng)人臍靜脈血管內(nèi)皮細(xì)胞(HUVECs),加入VEGF165,再加入不同濃度納米金,作用5 min,采用Western blot方法檢測(cè)細(xì)胞內(nèi)血管內(nèi)皮生長(zhǎng)因子受體-2下游信號(hào)磷酸化PLC-yl蛋白的水平。 結(jié)果1.血管內(nèi)皮細(xì)胞增殖抑制實(shí)驗(yàn)：納米金明顯抑制VEGF165誘導(dǎo)的血管內(nèi)皮細(xì)胞增殖作用,而對(duì)缺乏肝素結(jié)合位點(diǎn)的VEGF121沒(méi)有明顯抑制作用；2.AFM檢測(cè)到納米金與VEGF165、VEGF121作用前后血管內(nèi)皮細(xì)胞超微結(jié)構(gòu)變化,VEGF165誘導(dǎo)的血管內(nèi)皮細(xì)胞表面出現(xiàn)許多大小約數(shù)百納米的孔道或顆粒,細(xì)胞膜邊緣鋪展延伸。加入VEGF165、納米金后,細(xì)胞表面孔道或顆粒大為減少。VEGF121誘導(dǎo)的血管內(nèi)皮細(xì)胞在納米金加入前后的表面超微結(jié)構(gòu)無(wú)明顯變化；3.共聚焦顯微鏡和近場(chǎng)光學(xué)顯微鏡探測(cè)到納米金阻斷了VEGF165與其細(xì)胞膜表面受體VEGFR2的結(jié)合。4.VEGF165濃度不變(10μg/L),隨著納米金溶液濃度的增加(125、250、500nmol/L),納米金抑制PLC-γ1磷酸化作用越來(lái)越明顯,而對(duì)缺乏肝素結(jié)合位點(diǎn)的VEGF121沒(méi)有明顯抑制作用。 結(jié)論1.納米金抑制了VEGF165促血管內(nèi)皮細(xì)胞增殖的作用：2.納米金能夠阻止具有肝素結(jié)合位點(diǎn)的VEGF165與其受體VEGFR2的結(jié)合,阻止其下游的細(xì)胞信號(hào)傳導(dǎo),從而抑制了VEGF165誘導(dǎo)的血管內(nèi)皮細(xì)胞增殖：3.結(jié)合量子點(diǎn)標(biāo)記技術(shù),高分辨率的近場(chǎng)光學(xué)顯微鏡是研究生物單分子間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- 緯 _ 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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 丁暉;潘運(yùn)龍;覃莉;陳茜;;正常人臍靜脈內(nèi)皮細(xì)胞的原子力顯微鏡觀察[J];暨南大學(xué)學(xué)報(bào)(自然科學(xué)與醫(yī)學(xué)版);2011年02期

2 ;Atomic Force Microscopy-based Cell Nanostructure for Ligand-conjugated Quantum Dot Endocytosis[J];Acta Biochimica et Biophysica Sinica;2006年09期

3 潘運(yùn)龍;邱思遠(yuǎn);覃莉;王存川;丁暉;程欣;杜彬;;納米金與VEGF_(165)分子作用的原子力顯微鏡研究[J];中國(guó)病理生理雜志;2010年12期

4 段惠莉,李君文;納米顆粒在生物學(xué)檢測(cè)中的研究進(jìn)展[J];中國(guó)公共衛(wèi)生;2005年07期

5 潘運(yùn)龍;覃莉;蔡繼業(yè);;量子點(diǎn)在生物分子、細(xì)胞及體內(nèi)生物成像的研究進(jìn)展[J];中華實(shí)驗(yàn)外科雜志;2006年10期

，

本文編號(hào)：1545157