【作者】 房晓娜；

【导师】 韩炜； 宋宏伟；

【作者基本信息】 吉林大学 ， 应用物理， 2012， 博士

【摘要】 为了满足生物学领域飞速发展的技术需求，同时伴随着荧光材料和显微技术的逐渐发展，荧光技术已成为生物研究很多领域的重要技术手段。而近年来由于生物原位探测技术的发展使越来越多的研究者专注于生物体内探测，如利用荧光分子对生物细胞进行标记来研究细胞内某些组织的结构、利用荧光蛋白对某些基因进行标记来监测这些基因的表达情况等。由于生物体内原位检测具有更高的真实性和可信度，使得荧光技术在这方面的应用也越来越被人们所关注。要实现生物体内荧光探测，首先需要对靶向分子进行标记。目前被广泛应用的主要有两种形式的标签：一种是利用某些荧光材料与细胞体内靶向蛋白或DNA等分子特异性结合，通过对这些荧光分子的探测达到研究靶向分子的目的。而另一种是在DNA水平上利用荧光蛋白基因对表达目的蛋白的基因进行标记，通过检测荧光蛋白实现对其报告基因表达情况进行研究的目的。本论文分别对两种生物体荧光探针进行了研究。对于第一种形式的探索，鉴于目前的研究重点还集中寻找荧光特性比染料分子优异、生物毒性却比量子点低的荧光材料，而近年来有些研究者发现稀土配合物的许多优良的特性，使其成为一种非常有前景的生物荧光探测材料，同时由于生物体内所有生物分子及成份都是液相环境工作的，故我们对Eu配合物在溶液中的光学特性进行研究，同时为了提高其荧光探测灵敏度，我们也对溶液中Ag纳米粒子增强该Eu配合物荧光的机理进行了研究，为溶液中金属增强荧光技术提供理论依据。我们制备了不同浓度的Eu（TTA）₃·2H₂O配合物的二甲基甲酰胺（DMF）溶液，同时利用还原法制备了均匀分散的Ag纳米粒子胶体溶液，研究了Ag纳米粒子对配合物Eu（TTA）₃·2H₂O的DMF溶液发光的增强和猝灭的机理。我们通过吸收光谱、激发和发射光谱、发光动力学等性质的研究，揭示了Ag纳米粒子对Eu配合物荧光增强和猝灭的机制。实验结果显示，Ag纳米粒子对不同浓度的配合物溶液分别表现为增强和猝灭,当配合物浓度较高时，配合物荧光强度随Ag浓度增加先增强后减弱，此时配合物分子之间存在较强的相互作用，而Ag纳米粒子会降低这种相互作用。在溶液中，Ag纳米粒子可以通过增强Eu³⁺周围的局域场提高⁵D₀-⁷F₂的电偶极辐射跃迁速率；同时通过减小配合物分子间相互作用降低配合物分子间的荧光共振能量传递，从而降低⁵D0的非辐射跃迁速率。这两种因素导致稀土配合物荧光增强。另一方面，当配合物浓度较低时，Ag纳米粒子对配合物荧光表现为猝灭，这是Ag纳米粒子和配合物对激发光吸收竞争导致的。我们还研究了一定浓度的配合物Eu（TTA）₃·2H₂O的DMF溶液中，Ag纳米粒子对配合物的荧光增强效应与激发波长的依赖关系。我们分别选择269nm，341nm和375nm波长的光作为激发光，研究了不同激发波长下该Eu配合物溶液的激发和发射强度随溶液中Ag纳米粒子浓度的变化关系。通过对吸收光谱、激发和发射光谱等荧光性质的研究，我们发现Ag纳米粒子的存在不仅可以增强Eu³⁺的⁵D₀-⁷F₂的电偶极辐射跃迁速率，同时还可以提高激发效率，增强激发强度。不同波长激发时Ag纳米粒子对配合物溶液激发和发射强度增强的程度存在很大差异，当采用375nm激发时，荧光增强效果最好。同时实验结果还显示，Ag纳米粒子的存在还降低了Eu配合物内部有机配体TTA向发光中心Eu³⁺的能量传递效率。通过分析得出，Ag纳米粒子不仅可以增强Eu³⁺的辐射跃迁速率、降低溶液中配合物的非辐射跃迁速率，同时还可以增强局域场、提高配合物溶液的激发效率，降低配合物内部的能量传递效率，这些因素共同决定了溶液中配合物的荧光强度。而对于第二种形式，自荧光蛋白被发现以来，由于其无毒性、可以对靶向蛋白进行精确标记、可以标记活细胞并实时观测等其它荧光材料所不具备的优良特性，使其成为生物体内基因表达研究中不可替代的技术。而近年来由于人们通过对荧光蛋白进行改造得到了很多不同颜色、不同特性的荧光蛋白变体，这使得其应用更加广泛。本文中我们利用具有成熟快、荧光信号强的黄色荧光蛋白变体Venus作为报告基因，对自抑制调控回路中的调控蛋白的表达情况进行了研究。我们构建了以Venus作为报告子的tetR自抑制调控回路，并将该回路整合到大肠杆菌染色体上，实现回路的单拷贝。通过改变诱导剂aTc的浓度和诱导时间来达到改变自抑制回路中反应参数的目的，从而研究处于不同环境中不同参数下自抑制回路调控蛋白（即抑制子TetR）的表达分布。通过研究我们发现在蛋白分子与DNA结合和解离速率比降解速率快得多的时候，系统处于绝热近似的状态，此时所有细胞只有一种表达状态；而当我们同过调节诱导条件使得结合速率与解离速率与降解速率相当，甚至低于降解速率的时候，系统处于一种非绝热的状态，我们的结果显示，此时细胞不再是一种表达状态，而是存在两种可能的状态，即在同一环境的细胞群体中，部分细胞处于高表达水平，而另一部分细胞却处在低表达状态。该实验结果与我们小组通过建立自抑制回路的理论模型得到的模拟结果完全吻合，进一步说明了理论预言的正确性，以及该实验结果的可靠性。这一研究结果从根本上为细胞分化的研究提供了更加可靠地依据。更多还原

【Abstract】 In order to meet the technical requirements of the rapid development in biologyfiled,also accompanied by the gradually developed of the fluorescent material andmicroscopy,fluorescence technology has become a very important technique in manyareas of biological research.In recent years more and more researchers focued on thein vivo detection,such as labeling some specific tissues in the cells to study theirstructure,or labeling label a protein by a fluorescence protein to measure itsexpression level.Due to the higher authenticity and credibility of in situ detecion invivo than in vitro,more and more people began to pay attention to this area.The mostimportant technique of in situ detection was how to label the targeting molecules.Two kinds of lables are widely used now.One was that using the fluorscencemoecules specifically binding to the target,and studying the target molecules bydetecting the fluorescence labels.The other form of label was fluorescence protein.The protein labeled by a fluorescence protein（FP）was extremely specific,and theexpression level of target gene was reported and detected by the FP.Here wediscussed these two forms of fluorescence labelsFor the first form,the current key issue was still looking for a promisingcandidate fluorescence label，which had better optical properties than dye and lowertoxicity than Quantum Dot.Due to the excellent characteristics of the rare earthcomplexes,many researchers considered it as a very promising fluorescence materialfor in vivo detection.Because all the moelecules inside the cells were situated insolution environment,we need to find a rare earth complexe which had goodfluorescence properties.In order to do that,we synthesized Eu complexes,and studiedits optical characteristic in solution phase.We also tried to enhance the fluorescenceintensity of the complexes in solution by Ag nanoparticles,and discussed the originsof metal enhanced fluorescence in solution.Luminescence enhancement and quenching of Eu（TTA）₃·2H₂O complex in DMF（N,N-dimethylformamide）solution containing silver nanoparticles were observed, which depended on the concentrations of both europium complex and silvernanoparticles.Their origins were discussed based on absorption spectra,excitationand emission spectra,and luminescent decay dynamics.The results indicated thatwhen the concentration of Eu complex was high enough,strong interaction amongcomplex molecules occurred.The presence of Ag nanoparticles definitely decreasedthe interaction among complex molecules.In the solution containing Ag nanoparticlesthe electronic-dipole transition rate of5D0-⁷F₂increased due to enhanced local fieldsurrounding Eu³⁺ions,while the nonradiative transition rate from5D0decreasedowing to decreased resonant energy transfer among europium complex molecules.These two factors led to the luminescence enhancement of europium complex.As tothe luminescent quenching,was attributed to absorption competition between Agnanoparticles and europium complex at excited wavelength.We also studied the wavelength dependence of Ag nanoparticles enhanced Eucomplexes fluorescence in solution.We selected three different wavelengthes,269nm,341nm and375nm as the excitation light,and explored how the excitation andemission intensity vary with increasing of Ag nanoparticles concentration.When wedicussed the absorption spectra,excitation and emission spectra,we found that Agnanoparticles could not only increase the electronic-dipole transition rate of Eu³⁺,butalso increase the excitation intensity and excitation efficiency.We also found that boththe enhancement factor of excitation and emission light intensity were very differentwhen we used different excitation wavelength.The results also showed that thepresence of Ag nanoparticles also reduced the efficiency of energy transfer betweenEu³⁺and organic ligands（TTA）.Finally,we concluded that Ag nanoparticles not onlyincreased the radiative transition,decreased nonradiative transition,but also enhancedthe local field effect of Ag nanoparticles,increased excitation efficiency of thecomplex solution,reduced internal energy transfer efficiency of Eu complexemolecule.All these factors worked together,finally enhanced the fluorescence of Eucomplex in solution.For the fluorescence protein,since it also was a protein,so it did not have atoxic effect on cell.Meanwhile,the genetically encoded FPs were extremely specific when it was used to label a protein.These advantages made it became the mostsuitable fluorescence label in the in vivo detection since the GFP was discovered.Asfurther development of GFP and finding of its variants with different fluorescencebehaviour,the fluorescence protein has been used by more and more researchers indifferent biolodical field.We constructed a tetR self-repressor gene regulation circuit,and used Venus asthe reporter of transcription factor TetR.Here,Venus is a YFP variant whichmaturated very fast and had higher fluorescence signal than many other kindfluorescence proteins.In order to make sure the copy number of the self-repressorgene regulation circuit is single-copy,we integrated it on the chromosome of E.coli.We induced the cells under different concentration of aTc to change the biochemicalreaction parameters of the regutated circuit,such as the binding and unbinding ratesbetween TetR and oprator tetO.We explored the expression level distribution of TetRin different biochemical environment by monitoring fluorescence of Venus.Ourresults showed that when the binding/unbinding were much faster than thesynthesis/degradation of protein,the concentration distribution of TetR in all the cellsexhibited one peak under the data from flow cytometry.But when we tuned thecondition of inducing to make the the binding/unbinding were even slower than thesynthesis/degradation,we found that there were two peaks happened,which meantthat there were two population cells with different expression level of regulatorprotein even when the same kind of cells grew in same environment.This result fittedto the theoretical prediction which was also got in our group very well.It must be asignificant breakthrough in the research filed of cell differentiation.更多还原