本文選題：沒食子酸　切入點：金納米顆粒　出處：《吉林大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：沒食子酸（gallic acid, GA）或3,4,5-三羥基苯甲酸，是一種天然的多酚類化合物，作為自由分子或單寧酸分子的一部分，廣泛存在于水果和植物中，具有天然的抗氧化、抗過敏、抗誘變、抗癌、抗病毒、抗菌和抗炎等生物活性。經(jīng)研究證實，GA可誘導(dǎo)多種腫瘤細(xì)胞凋亡，在預(yù)防癌癥轉(zhuǎn)移和惡化中起到非常重要的作用。 納米材料是指那些在三維空間中有一維或多維處于納米尺度（1-100nm）范圍內(nèi)的材料。其本身及由它們作為基本單元構(gòu)成的新材料，由于小尺寸和大比表面積等效應(yīng)而有獨特的光學(xué)、電子或機械性能。具有卓越物理和化學(xué)性質(zhì)的納米材料正日益滲入到生物和醫(yī)學(xué)研究領(lǐng)域，為現(xiàn)代生物學(xué)成像，醫(yī)學(xué)診斷和治療提供了更加靈敏和可靠的材料和方法。金、銀等貴金屬納米顆粒或者以其為結(jié)構(gòu)單元所構(gòu)成的納米材料在光學(xué)、催化、生物工程和醫(yī)學(xué)等研究領(lǐng)域占有非常重要的地位。 GA作為還原劑和穩(wěn)定劑合成的金納米顆粒（gold nanoparticles, AuNPs）曾被用于生物分析，但此種金納米顆粒對人類細(xì)胞系毒性還沒有被研究過。我們首先以GA同時作為還原劑和穩(wěn)定劑，用簡單、“綠色”的一步法合成了GA修飾的金納米顆粒（GA-AuNPs），并對其吸收光譜和尺寸等進(jìn)行了表征。通過比較合成反應(yīng)前后的溶液中GA的紫外吸收來確定結(jié)合于GA-AuNPs上GA的摩爾數(shù)。通過與等摩爾數(shù)的自由GA生物活性比較，確定了含有終濃度為0.065，0.13，0.26，0.52和0.65mM結(jié)合GA的GA-AuNPs對人類肝細(xì)胞（humanhepatocytecell, HL-7702）的生長和凋亡的影響。實驗結(jié)果表明：含有相同摩爾數(shù)結(jié)合GA的GA-AuNPs的細(xì)胞毒性和細(xì)胞凋亡的誘導(dǎo)能力與自由GA相比均有很大程度的下降。GA-AuNPs合成反應(yīng)過程中，GA所發(fā)生的主要化學(xué)變化是其分子結(jié)構(gòu)中的兩個酚羥基被氧化成其相應(yīng)的醌形式。我們推斷GA分子的毒性與它的酚羥基有很大關(guān)系，即GA酚羥基是決定其生物活性的主要基團。這與前人的用其它方法所取得的結(jié)論一致。因此，本研究在驗證了GA-AuNPs細(xì)胞毒性和對細(xì)胞凋亡影響情況的同時，也發(fā)現(xiàn)了一個簡單的新方法來證明GA的酚羥基對生物活性具有決定性作用。 微生物對抗生素的耐藥性為抗微生物感染新藥的開發(fā)帶來了巨大挑戰(zhàn)。銀納米顆粒（silver nanoparticles, AgNPs）因其廣譜的抗菌活性和使用后不會產(chǎn)生新的耐藥菌株等特性，已逐步成為非常有吸引力的抗生素替代產(chǎn)品。我們以一個簡單、“綠色”的方法合成了GA修飾的銀納米顆粒（GA-AgNPs），其中GA同時作為GA-AgNPs的還原劑和穩(wěn)定劑。銀納米顆粒形態(tài)、尺寸、zeta電勢和吸收光譜分別用透射電子顯微鏡，動態(tài)光散射和紫外可見光譜進(jìn)行了表征。我們用微量稀釋等方法分別測得GA-AgNPs對三種微生物：大腸桿菌（Escherichiacoli, E. coli）、金黃葡萄球菌（Staphylococcus aureus, S. aureus）和白色念珠菌（Candida albicans, C. albicans）的最小抑菌濃度（Minimal InhibitoryConcentration, MICs）分別為6、30和24μg/mL。然后，我們用MTT法和Calcein-AM/PI染色法分別考察了當(dāng)終濃度為6，12，18，24和30μg/mL時，GA-AgNPs對正常肝細(xì)胞（HL-7702）和宮頸癌細(xì)胞（human cervical carcinoma,HeLa）的毒性。研究結(jié)果表明GA-AgNPs在濃度大于24μg/mL時，其對HeLa細(xì)胞的毒性明顯高于對照組（P 0.05）；而當(dāng)其終濃度為30μg/mL時，GA-AgNPs對HL-7702細(xì)胞的毒性與對照組相比沒有明顯差異。接著我們采用Annexin-V/PI結(jié)合和Hoechst33342的核染色分析兩種分析方法，分別比較了GA-AgNPs對HeLa和HL-7702細(xì)胞凋亡的影響。實驗結(jié)果顯示，經(jīng)過上述濃度的GA-AgNPs處理后，HeLa細(xì)胞組的無論是早期還是晚期凋亡細(xì)胞的比例均高于HL-7702細(xì)胞組，，而且相對活細(xì)胞比例分析的結(jié)果與毒性分析的結(jié)果一致。最后，我們對GA-AgNPs的細(xì)胞毒性和細(xì)胞凋亡的機理進(jìn)行研究，我們分別測試了兩種細(xì)胞經(jīng)過GA-AgNPs處理后的線粒體膜電位（mitochondrialmembrane potential, MMP）變化和細(xì)胞內(nèi)活性氧（reactive oxygen species, ROS）的產(chǎn)生情況。實驗結(jié)果證明，GA-AgNPs引起了細(xì)胞內(nèi)MMP降低和ROS的產(chǎn)生，特別是在HeLa細(xì)胞中，這兩種變化較為明顯。綜上所述，GA-AgNPs主要是通過降低細(xì)胞內(nèi)MMP和引起細(xì)胞內(nèi)ROS的產(chǎn)生來誘導(dǎo)HeLa細(xì)胞的凋亡以及壞死。基于抗菌和選擇性癌細(xì)胞毒性的特殊性質(zhì)，GA-AgNPs有望成為可用于醫(yī)學(xué)領(lǐng)域的新型廣譜抗菌納米材料。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R114

【參考文獻(xiàn)】

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1 劉作家;沒食子酸與人血清白蛋白相互作用及其誘導(dǎo)胰腺癌細(xì)胞凋亡的機制研究[D];吉林大學(xué);2013年

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