没食子酸修饰金、银纳米颗粒的细胞毒性研究

发布时间：2018-03-23 05:36

本文选题：没食子酸　切入点：金纳米颗粒　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：没食子酸（gallic acid, GA）或3,4,5-三羟基苯甲酸，是一种天然的多酚类化合物，作为自由分子或单宁酸分子的一部分，广泛存在于水果和植物中，具有天然的抗氧化、抗过敏、抗诱变、抗癌、抗病毒、抗菌和抗炎等生物活性。经研究证实，GA可诱导多种肿瘤细胞凋亡，在预防癌症转移和恶化中起到非常重要的作用。纳米材料是指那些在三维空间中有一维或多维处于纳米尺度（1-100nm）范围内的材料。其本身及由它们作为基本单元构成的新材料，由于小尺寸和大比表面积等效应而有独特的光学、电子或机械性能。具有卓越物理和化学性质的纳米材料正日益渗入到生物和医学研究领域，为现代生物学成像，医学诊断和治疗提供了更加灵敏和可靠的材料和方法。金、银等贵金属纳米颗粒或者以其为结构单元所构成的纳米材料在光学、催化、生物工程和医学等研究领域占有非常重要的地位。 GA作为还原剂和稳定剂合成的金纳米颗粒（gold nanoparticles, AuNPs）曾被用于生物分析，但此种金纳米颗粒对人类细胞系毒性还没有被研究过。我们首先以GA同时作为还原剂和稳定剂，用简单、“绿色”的一步法合成了GA修饰的金纳米颗粒（GA-AuNPs），并对其吸收光谱和尺寸等进行了表征。通过比较合成反应前后的溶液中GA的紫外吸收来确定结合于GA-AuNPs上GA的摩尔数。通过与等摩尔数的自由GA生物活性比较，确定了含有终浓度为0.065，0.13，0.26，0.52和0.65mM结合GA的GA-AuNPs对人类肝细胞（humanhepatocytecell, HL-7702）的生长和凋亡的影响。实验结果表明：含有相同摩尔数结合GA的GA-AuNPs的细胞毒性和细胞凋亡的诱导能力与自由GA相比均有很大程度的下降。GA-AuNPs合成反应过程中，GA所发生的主要化学变化是其分子结构中的两个酚羟基被氧化成其相应的醌形式。我们推断GA分子的毒性与它的酚羟基有很大关系，即GA酚羟基是决定其生物活性的主要基团。这与前人的用其它方法所取得的结论一致。因此，本研究在验证了GA-AuNPs细胞毒性和对细胞凋亡影响情况的同时，也发现了一个简单的新方法来证明GA的酚羟基对生物活性具有决定性作用。微生物对抗生素的耐药性为抗微生物感染新药的开发带来了巨大挑战。银纳米颗粒（silver nanoparticles, AgNPs）因其广谱的抗菌活性和使用后不会产生新的耐药菌株等特性，已逐步成为非常有吸引力的抗生素替代产品。我们以一个简单、“绿色”的方法合成了GA修饰的银纳米颗粒（GA-AgNPs），其中GA同时作为GA-AgNPs的还原剂和稳定剂。银纳米颗粒形态、尺寸、zeta电势和吸收光谱分别用透射电子显微镜，动态光散射和紫外可见光谱进行了表征。我们用微量稀释等方法分别测得GA-AgNPs对三种微生物：大肠杆菌（Escherichiacoli, E. coli）、金黄葡萄球菌（Staphylococcus aureus, S. aureus）和白色念珠菌（Candida albicans, C. albicans）的最小抑菌浓度（Minimal InhibitoryConcentration, MICs）分别为6、30和24μg/mL。然后，我们用MTT法和Calcein-AM/PI染色法分别考察了当终浓度为6，12，18，24和30μg/mL时，GA-AgNPs对正常肝细胞（HL-7702）和宫颈癌细胞（human cervical carcinoma,HeLa）的毒性。研究结果表明GA-AgNPs在浓度大于24μg/mL时，其对HeLa细胞的毒性明显高于对照组（P 0.05）；而当其终浓度为30μg/mL时，GA-AgNPs对HL-7702细胞的毒性与对照组相比没有明显差异。接着我们采用Annexin-V/PI结合和Hoechst33342的核染色分析两种分析方法，分别比较了GA-AgNPs对HeLa和HL-7702细胞凋亡的影响。实验结果显示，经过上述浓度的GA-AgNPs处理后，HeLa细胞组的无论是早期还是晚期凋亡细胞的比例均高于HL-7702细胞组，，而且相对活细胞比例分析的结果与毒性分析的结果一致。最后，我们对GA-AgNPs的细胞毒性和细胞凋亡的机理进行研究，我们分别测试了两种细胞经过GA-AgNPs处理后的线粒体膜电位（mitochondrialmembrane potential, MMP）变化和细胞内活性氧（reactive oxygen species, ROS）的产生情况。实验结果证明，GA-AgNPs引起了细胞内MMP降低和ROS的产生，特别是在HeLa细胞中，这两种变化较为明显。综上所述，GA-AgNPs主要是通过降低细胞内MMP和引起细胞内ROS的产生来诱导HeLa细胞的凋亡以及坏死。基于抗菌和选择性癌细胞毒性的特殊性质，GA-AgNPs有望成为可用于医学领域的新型广谱抗菌纳米材料。
[Abstract]:Gallic acid (gallic acid GA) or 3,4,5- three hydroxy benzoic acid, is a natural polyphenolic compound, as a part of the free molecular or tannic acid molecules, widely exist in fruits and plants, has a natural antioxidant, anti allergic, anti mutation, anti-cancer, anti viral, anti-inflammatory and antibacterial activity. The study confirmed that GA can induce apoptosis in various tumor cells, plays a very important role in the prevention of cancer metastasis and progression.
Nano materials are those in three-dimensional space is one-dimensional or multidimensional in nanometer scale (1-100nm) within the scope of the material itself. And by them as a basic element of the new material, due to the small size and large surface area effect and unique optical, electronic or mechanical energy. Nano material has excellent physical and the chemical properties are increasingly infiltrated into the field of biological and medical research, modern biological imaging, provides more sensitive and reliable materials and methods of medical diagnosis and treatment. Gold, silver and other noble metal nanoparticles or as the nano material structure of single element in optical, catalytic, plays a very important role in biological engineering and medical research.
GA as the gold nanoparticles were synthesized by reducing agent and stabilizer (gold nanoparticles AuNPs) has been used for biological analysis, but the toxicity of gold nanoparticles on human cell lines have not been studied. We first to GA at the same time as the simple agent and stabilizer, reduction, "gold nanoparticle modified GA one-step process green" (GA-AuNPs), and the absorption spectrum and size were characterized. The solution before and after reaction of GA in UV absorption to determine the number of moles of binding to GA-AuNPs GA. The biological activity of free GA and equimolar number are determined, with the final concentration of 0.065,0.13,0.26,0.52 and 0.65mM combined with GA GA-AuNPs on human liver cells (humanhepatocytecell, HL-7702) the effect of growth and apoptosis. The experimental results show that with the same molar with GA and cytotoxicity of GA-AuNPs cells. The death inducing ability compared with the free GA are greatly decreased.GA-AuNPs synthesis in the reaction process, the main chemical changes of GA occurred two phenolic hydroxyl groups in the molecular structure is oxidized to the corresponding quinone form. We conclude that GA molecular toxicity has great relationship with the phenolic hydroxyl of it, namely GA phenol the hydroxyls are the key to determine its biological activity. This conforms with the previous conclusions obtained by other methods. Therefore, this study is to verify GA-AuNPs cell cytotoxicity and at the same time on cell apoptosis, phenolic hydroxyl group also found a simple new method to prove GA has a decisive effect on the biological activity.
Microbial resistance to antibiotics for anti infection drug development of microorganisms has brought great challenges. Silver nanoparticles (silver nanoparticles AgNPs) because of its broad-spectrum antibacterial activity and after use will not produce new resistant strains and other characteristics, has gradually become a very attractive alternative to antibiotics. We use a simple, "silver nanoparticles GA particles modified by the synthesis of green" method (GA-AgNPs), where GA and GA-AgNPs as reducing agent and stabilizer. The silver nano particle morphology, size, zeta potential and absorption spectra were characterized by transmission electron microscopy, dynamic light scattering and UV visible spectra were characterized by micro dilution method. We measured respectively. GA-AgNPs of three kinds of microorganisms: Escherichia coli (Escherichiacoli, E., coli), Staphylococcus aureus (Staphylococcus aureus, S. aureus) and Candida albicans (Candida albicans, C. albicans) the minimum inhibitory concentration (Minimal InhibitoryConcentration, MICs) were 6,30 and 24 g/mL.. Then we use the MTT method and Calcein-AM/PI staining method were investigated when the final concentration was 6,12,18,24 and 30 g/mL, GA-AgNPs of normal liver cells (HL-7702) and cervical cancer cells (human cervical carcinoma, HeLa). Study on the toxicity. The results show that the GA-AgNPs concentration is greater than 24 g/mL, the toxicity of HeLa cells was significantly higher than the control group (P 0.05); and when the final concentration was 30 g/mL, GA-AgNPs toxicity in HL-7702 cells compared with the control group had no obvious difference. Then we use the analysis of the two methods of nuclear staining combination of Annexin-V/PI and Hoechst33342, respectively, compared the effect of GA-AgNPs on HeLa and HL-7702 cell apoptosis. The experimental results show that the concentration of GA-AgNPs after treatment, HeLa cell groups whether early Is a late percentage of apoptotic cells was higher than that of HL-7702 cells, and analysis of relative proportion of living cells results and toxicity results. Finally, we studied the mechanism of GA-AgNPs cell toxicity and apoptosis, we tested two kinds of cells through the mitochondrial membrane potential after treatment with GA-AgNPs (mitochondrialmembrane potential MMP) the changes of active oxygen species and cells (reactive oxygen species, ROS). The experimental results show that GA-AgNPs caused the decrease of intracellular MMP and ROS, especially in HeLa cell, the two kinds of changes are obvious. In conclusion, GA-AgNPs is mainly caused by the ROS cells to produce HeLa cells induced by to reduce the intracellular MMP and apoptosis and necrosis. The special properties of antibacterial and selective cancer cell toxicity based on GA-AgNPs, is expected to become a new type can be used in the field of medicine. Broad-spectrum antibacterial nanomaterials.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R114

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