熒光金納米團(tuán)簇用于病毒成像分析及抗病毒研究
發(fā)布時(shí)間：2019-06-29 16:11
【摘要】：熒光金納米團(tuán)簇(Au NCs)因其亞納米級(jí)尺寸表現(xiàn)出獨(dú)特的光電化學(xué)性質(zhì),如光穩(wěn)定性好、雙光子吸收截面積大和良好的生物相容性等優(yōu)點(diǎn),在離子檢測(cè)、生物分子檢測(cè)、細(xì)胞成像、活體診斷和抗腫瘤方面都有研究報(bào)道,然而AuNCs對(duì)病毒的影響鮮有報(bào)道。本論文探究不同修飾劑的AuNCs對(duì)豬偽狂犬病毒(PRV)及豬繁殖與呼吸系統(tǒng)綜合征病毒(PRRSV)的影響,并將其進(jìn)行細(xì)胞成像研究。1.以谷胱甘肽(GSH)為修飾劑合成水溶性的熒光GSH-AuNCs,采用熒光光譜(FL)、紫外光譜(UV)、水合粒徑(DLS)、透射電子顯微鏡(TEM)、傅里葉變換紅外光譜(FT-IR)和X-射線光電子能譜(XPS)對(duì)GSH-Au NCs進(jìn)行表征,用MTT法評(píng)估了GSH-Au NCs在非洲綠猴腎傳代細(xì)胞(MARC-145)和豬腎傳代細(xì)胞系(PK-15)的細(xì)胞毒性,結(jié)果表明:GSH-Au NCs具有良好的光穩(wěn)定性,當(dāng)其濃度小于450 μmol/L時(shí),PK-15和MARC-145細(xì)胞的存活率大于80%。將GSH-Au NCs應(yīng)用于PRV入侵PK-15細(xì)胞和PRRSV入侵MARC-145細(xì)胞的影響研究,通過(guò)空斑試驗(yàn)、間接免疫熒光試驗(yàn)和Weston blot試驗(yàn),結(jié)果表明:GSH-Au NCs具有選擇性抗PRRSV的作用,而對(duì)PRV無(wú)明顯影響。最后以GSH-Au NCs為熒光探針通過(guò)激光共聚焦掃描顯微鏡觀察PRV入侵PK-15細(xì)胞的過(guò)程,觀察到PRV侵入PK-15細(xì)胞12 h后在細(xì)胞核發(fā)現(xiàn)明顯的綠色熒光,說(shuō)明PRV在復(fù)制后期會(huì)明顯的破壞PK-15細(xì)胞的細(xì)胞核。這將為AuNCs應(yīng)用于抗病毒領(lǐng)域提供一定的借鑒意義。2.以牛血清白蛋白(BSA)為修飾劑合成水溶性的熒光BSA-Au NCs和Au-Ag NCs,并對(duì)合成的AuNCs進(jìn)行FL、UV、DLS和FT-IR的表征,用MTT法評(píng)估了BSA-Au NCs和Au-Ag NCs在PK-15細(xì)胞的細(xì)胞毒性,結(jié)果表明:BSA-Au NCs和Au-Ag NCs具有良好的光穩(wěn)定性,當(dāng)兩種Au NCs的濃度小于600 μmol/L時(shí),PK-15細(xì)胞的存活率在80%以上。將BSA-Au NCs和Au-Ag NCs用于PK-15細(xì)胞成像研究,發(fā)現(xiàn)這兩種Au NCs在PK-15細(xì)胞中觀察不到明顯的熒光。最后,探究這兩種Au NCs對(duì)于PRV入侵PK-15細(xì)胞的影響,發(fā)現(xiàn)BSA-Au NCs和Au-Ag NCs對(duì)于PRV入侵PK-15細(xì)胞無(wú)明顯的影響。這一研究結(jié)果有望將AuNCs進(jìn)一步應(yīng)用于病毒的標(biāo)記研究。
[Abstract]:Fluorescent gold nanoclusters (AuNCs) have been reported in ion detection, biomolecule detection, cell imaging, in vivo diagnosis and antitumor because of their unique photoelectrochemical properties, such as good photostability, large two-photonic absorption cross section and good biocompatibility. However, the effect of AuNCs on virus is rarely reported. In this paper, the effects of AuNCs with different modifiers on Porcine pseudorabies virus (PRV) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) were studied. Water-soluble fluorescent GSH-AuNCs, was synthesized with glutathione (GSH) as modifier. GSH-AuNCs was characterized by fluorescence spectrum (FL), UV spectrum (UV), hydration particle size (DLS), transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The cytotoxicity of GSH-Au NCs in African green monkey kidney passage cells (MARC-145) and pig kidney passage cell lines (PK-15) was evaluated by MTT assay. The results showed that GSH-Au NCs had good photostability. When the concentration of GSH-Au NCs was less than 450 渭 mol/L, the survival rate of PK-15 and MARC-145 cells was more than 80%. GSH-Au NCs was applied to the study of the effect of PRV invading PK-15 cells and PRRSV invading MARC-145 cells. The results of plaque test, indirect immunofluorescence test and Weston blot test showed that GSH-Au NCs had selective anti-PRRSV effect, but had no obvious effect on PRV. Finally, GSH-Au NCs was used as fluorescence probe to observe the process of PRV invading PK-15 cells by laser confocal scanning microscope. The obvious green fluorescence was found in the nucleus of PK-15 cells after PRV invaded PK-15 cells for 12 hours, which indicated that PRV could obviously destroy the nucleus of PK-15 cells at the later stage of replication. This will provide a certain reference for the application of AuNCs in the field of antivirus. 2. Water soluble fluorescent BSA-Au NCs and Au-Ag NCs, were synthesized with bovine serum albumin (BSA) as modifiers, and the synthesized AuNCs was characterized by FL,UV,DLS and FT-IR. The cytotoxicity of BSA-Au NCs and Au-Ag NCs in PK-15 cells was evaluated by MTT method. The results showed that BSA-Au NCs and Au-Ag NCs had good photostability when the concentration of the two AuNCs was less than 600 渭 mol/L. The survival rate of PK-15 cells was more than 80%. BSA-Au NCs and Au-Ag NCs were used to study the imaging of PK-15 cells. It was found that the two kinds of Au NCs had no obvious fluorescence in PK-15 cells. Finally, the effects of these two kinds of Au NCs on PRV invading PK-15 cells were investigated, and it was found that BSA-Au NCs and Au-Ag NCs had no significant effect on PRV invading PK-15 cells. This study is expected to further apply AuNCs to the study of virus markers.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TB383.1;S432.41
本文編號(hào)：2507946
[Abstract]:Fluorescent gold nanoclusters (AuNCs) have been reported in ion detection, biomolecule detection, cell imaging, in vivo diagnosis and antitumor because of their unique photoelectrochemical properties, such as good photostability, large two-photonic absorption cross section and good biocompatibility. However, the effect of AuNCs on virus is rarely reported. In this paper, the effects of AuNCs with different modifiers on Porcine pseudorabies virus (PRV) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) were studied. Water-soluble fluorescent GSH-AuNCs, was synthesized with glutathione (GSH) as modifier. GSH-AuNCs was characterized by fluorescence spectrum (FL), UV spectrum (UV), hydration particle size (DLS), transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The cytotoxicity of GSH-Au NCs in African green monkey kidney passage cells (MARC-145) and pig kidney passage cell lines (PK-15) was evaluated by MTT assay. The results showed that GSH-Au NCs had good photostability. When the concentration of GSH-Au NCs was less than 450 渭 mol/L, the survival rate of PK-15 and MARC-145 cells was more than 80%. GSH-Au NCs was applied to the study of the effect of PRV invading PK-15 cells and PRRSV invading MARC-145 cells. The results of plaque test, indirect immunofluorescence test and Weston blot test showed that GSH-Au NCs had selective anti-PRRSV effect, but had no obvious effect on PRV. Finally, GSH-Au NCs was used as fluorescence probe to observe the process of PRV invading PK-15 cells by laser confocal scanning microscope. The obvious green fluorescence was found in the nucleus of PK-15 cells after PRV invaded PK-15 cells for 12 hours, which indicated that PRV could obviously destroy the nucleus of PK-15 cells at the later stage of replication. This will provide a certain reference for the application of AuNCs in the field of antivirus. 2. Water soluble fluorescent BSA-Au NCs and Au-Ag NCs, were synthesized with bovine serum albumin (BSA) as modifiers, and the synthesized AuNCs was characterized by FL,UV,DLS and FT-IR. The cytotoxicity of BSA-Au NCs and Au-Ag NCs in PK-15 cells was evaluated by MTT method. The results showed that BSA-Au NCs and Au-Ag NCs had good photostability when the concentration of the two AuNCs was less than 600 渭 mol/L. The survival rate of PK-15 cells was more than 80%. BSA-Au NCs and Au-Ag NCs were used to study the imaging of PK-15 cells. It was found that the two kinds of Au NCs had no obvious fluorescence in PK-15 cells. Finally, the effects of these two kinds of Au NCs on PRV invading PK-15 cells were investigated, and it was found that BSA-Au NCs and Au-Ag NCs had no significant effect on PRV invading PK-15 cells. This study is expected to further apply AuNCs to the study of virus markers.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TB383.1;S432.41
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 楊群峰;劉建云;陳華萍;王顯祥;黃乾明;單志;;貴金屬納米團(tuán)簇的制備及在生物檢測(cè)中的應(yīng)用[J];化學(xué)進(jìn)展;2011年05期
，本文編號(hào)：2507946
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