荧光金纳米团簇用于病毒成像分析及抗病毒研究
发布时间：2019-06-29 16:11
【摘要】：荧光金纳米团簇(Au NCs)因其亚纳米级尺寸表现出独特的光电化学性质,如光稳定性好、双光子吸收截面积大和良好的生物相容性等优点,在离子检测、生物分子检测、细胞成像、活体诊断和抗肿瘤方面都有研究报道,然而AuNCs对病毒的影响鲜有报道。本论文探究不同修饰剂的AuNCs对猪伪狂犬病毒(PRV)及猪繁殖与呼吸系统综合征病毒(PRRSV)的影响,并将其进行细胞成像研究。1.以谷胱甘肽(GSH)为修饰剂合成水溶性的荧光GSH-AuNCs,采用荧光光谱(FL)、紫外光谱(UV)、水合粒径(DLS)、透射电子显微镜(TEM)、傅里叶变换红外光谱(FT-IR)和X-射线光电子能谱(XPS)对GSH-Au NCs进行表征,用MTT法评估了GSH-Au NCs在非洲绿猴肾传代细胞(MARC-145)和猪肾传代细胞系(PK-15)的细胞毒性,结果表明:GSH-Au NCs具有良好的光稳定性,当其浓度小于450 μmol/L时,PK-15和MARC-145细胞的存活率大于80%。将GSH-Au NCs应用于PRV入侵PK-15细胞和PRRSV入侵MARC-145细胞的影响研究,通过空斑试验、间接免疫荧光试验和Weston blot试验,结果表明:GSH-Au NCs具有选择性抗PRRSV的作用,而对PRV无明显影响。最后以GSH-Au NCs为荧光探针通过激光共聚焦扫描显微镜观察PRV入侵PK-15细胞的过程,观察到PRV侵入PK-15细胞12 h后在细胞核发现明显的绿色荧光,说明PRV在复制后期会明显的破坏PK-15细胞的细胞核。这将为AuNCs应用于抗病毒领域提供一定的借鉴意义。2.以牛血清白蛋白(BSA)为修饰剂合成水溶性的荧光BSA-Au NCs和Au-Ag NCs,并对合成的AuNCs进行FL、UV、DLS和FT-IR的表征,用MTT法评估了BSA-Au NCs和Au-Ag NCs在PK-15细胞的细胞毒性,结果表明:BSA-Au NCs和Au-Ag NCs具有良好的光稳定性,当两种Au NCs的浓度小于600 μmol/L时,PK-15细胞的存活率在80%以上。将BSA-Au NCs和Au-Ag NCs用于PK-15细胞成像研究,发现这两种Au NCs在PK-15细胞中观察不到明显的荧光。最后,探究这两种Au NCs对于PRV入侵PK-15细胞的影响,发现BSA-Au NCs和Au-Ag NCs对于PRV入侵PK-15细胞无明显的影响。这一研究结果有望将AuNCs进一步应用于病毒的标记研究。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TB383.1;S432.41
本文编号：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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TB383.1;S432.41
【参考文献】
相关期刊论文 前1条
1 杨群峰;刘建云;陈华萍;王显祥;黄乾明;单志;;贵金属纳米团簇的制备及在生物检测中的应用[J];化学进展;2011年05期
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