本文選題：腫瘤　切入點(diǎn)：乏氧　出處：《河北大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：乏氧是實體腫瘤微環(huán)境的重要特征之一，導(dǎo)致實體腫瘤對放、化療產(chǎn)生較強(qiáng)的抗性，是惡性腫瘤局部復(fù)發(fā)和惡性轉(zhuǎn)移的危害因素。因此對乏氧區(qū)域的檢測具有非常重要的意義。乏氧熒光成像法有較高的靈敏度，因此成為人們研究的熱點(diǎn)。 3-羥基黃酮衍生物具有較大的斯托克斯位移值、良好的光穩(wěn)定性、較高的量子產(chǎn)率等光學(xué)特性，是一類應(yīng)用比較廣泛的熒光物質(zhì)。本文以3-羥基黃酮類衍生物為發(fā)色團(tuán)，以硝基苯基和硝基咪唑基為乏氧響應(yīng)基團(tuán)，設(shè)計合成了系列乏氧熒光探針。因硝基的強(qiáng)淬滅作用，導(dǎo)致熒光分子熒光減弱。在乏氧環(huán)境下，，硝基化合物透過乏氧細(xì)胞脂膜，在硝基還原酶和煙酰胺腺嘌呤二核苷酸的作用下，硝基被還原為氨基，通過電子轉(zhuǎn)移反應(yīng)，響應(yīng)基團(tuán)與熒光分子發(fā)生斷裂，釋放出熒光分子，從而實現(xiàn)對乏氧的檢測。但是，硝基乏氧響應(yīng)基團(tuán)的引入，導(dǎo)致探針的水溶性大大降低。為此，在3-羥基黃酮分子的6位和8位引入嗎啉基側(cè)鏈，來增大其水溶性。采用NMR，HRMS和IR等波譜技術(shù)對探針的結(jié)構(gòu)進(jìn)行了表征，利用循環(huán)伏安法測定了探針的氧化還原電位，利用HRMS研究了乏氧探針的檢測機(jī)理，利用熒光光譜法探討了緩沖溶液種類、pH值、溶劑性質(zhì)和含量等因素對發(fā)色團(tuán)熒光性質(zhì)的影響，采用MTT法對探針的毒性進(jìn)行了評價，并采用熒光成像法將其應(yīng)用于乏氧細(xì)胞的檢測。 結(jié)果表明：乏氧響應(yīng)基團(tuán)和取代基的結(jié)構(gòu)影響探針的半波還原電位，3-（1’-甲基-4’-硝基咪唑）甲氧基-6,8-二嗎啉基甲基黃酮的半波還原電位（-0.802V）高于3-（4-硝基苯基）甲氧基-6,8-二嗎啉基甲基黃酮其半波還原電位（-0.812V），硝基咪唑修飾的探針更易被還原。4-（4＇-二甲氨基）苯基-3-羥基黃酮在6位和8位引入嗎啉基后，熒光發(fā)射波長由545nm紅移至554nm。熒光分子的熒光強(qiáng)度隨pH的增加而增大，4-（4＇-二甲氨基）苯基-6,8-二嗎啉基甲基-3-羥基黃酮在pH為7.8溶液中的熒光強(qiáng)度是在pH為5.0溶液中熒光強(qiáng)度的5倍；熒光分子的熒光強(qiáng)度隨助溶劑極性的增加而減小，4-（4＇-二甲氨基）苯基-6,8-二嗎啉基甲基-3-羥基黃酮在THF中的熒光強(qiáng)度高于在DMSO中；熒光分子的熒光強(qiáng)度隨助溶劑量的增加而增大。將濃度為10μmol/L的探針作用于Hela細(xì)胞24h后沒有表現(xiàn)出毒性；在乏氧細(xì)胞中，探針被還原，釋放出熒光分子，顯示出乏氧選擇性，可用于乏氧細(xì)胞的檢測。
[Abstract]:Hypoxia is one of the important characteristics of solid tumor microenvironment, which leads to strong resistance of solid tumor to radiotherapy and chemotherapy. It is a harmful factor for local recurrence and malignant metastasis of malignant tumor. Therefore, it is of great significance to detect the hypoxic region. Hypoxia fluorescence imaging has a high sensitivity, so it has become a hot research topic. The 3-hydroxyflavone derivatives are a kind of widely used fluorescent substances with large Stokes shift value, good light stability, high quantum yield and other optical properties. In this paper, the chromogenic group of 3-hydroxyflavonoids derivatives is used as chromogenic group. A series of anoxic fluorescence probes were designed and synthesized using nitrophenyl and nitroimidazolyl as hypoxia responsive groups. The fluorescence of fluorescence molecules weakened due to the strong quenching of nitro. In hypoxic environment, nitro compounds passed through the lipid membrane of hypoxic cells. In the presence of nitro reductase and nicotinamide adenine dinucleotide, the nitro group is reduced to amino group. By electron transfer reaction, the responsive group breaks with the fluorescent molecule and the fluorescent molecule is released. The water solubility of the probe was greatly reduced with the introduction of nitro-hypoxia responsive groups. Therefore, the morpholine side chain was introduced at 6 and 8 positions of 3-hydroxyflavone molecule. The structure of the probe was characterized by NMR-HRMS and IR spectroscopy, the redox potential of the probe was measured by cyclic voltammetry, and the detection mechanism of the probe was studied by HRMS. The effects of pH value of buffer solution, solvent property and content on the fluorescence properties of chromophore were studied by fluorescence spectrometry. The toxicity of the probe was evaluated by MTT method. Fluorescence imaging was used to detect hypoxia cells. The results showed that the half-wave reduction potential of the probe was influenced by the structure of hypoxic responsive groups and substituents. The half-wave reduction potential of the probe was higher than that of 3-O4-nitrophenyl) methoxy-8-dimorpholinyl methyl flavonoids (-0.802V). The half-wave reduction potential of -6 and 8-dimorpholinyl methyl flavonoids was -0.812V, and the probe modified by nitroimidazole was easier to be reduced by introducing morpholine group into phenyl-3-hydroxy flavonoids at the 6th and 8th positions, and the probe modified by nitroimidazole was more likely to be reduced to phenyl-3-hydroxy flavonoids at the 6 and 8 sites. The fluorescence emission wavelength shifted from 545 nm to 554 nm. The fluorescence intensity of the fluorescence molecule increased with the increase of pH. The fluorescence intensity of the fluorescence molecule in the solution of pH 7.8 was 5 times higher than that in the solution of pH 5.0. The fluorescence intensity of the fluorescence molecule decreased with the increase of the polarity of the cosolvent. The fluorescence intensity in THF was higher than that in DMSO. The fluorescence intensity of the fluorescent molecule increased with the increase of the amount of cosolvent. The probe with the concentration of 10 渭 mol/L was not toxic to Hela cells after 24 hours, and the probe was reduced to release fluorescent molecules in hypoxic cells, which showed hypoxia selectivity. It can be used for the detection of hypoxia cells.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R914;O657.3

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