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  • 學位論文

發展用於MCF-7細胞之具3-羥基黃酮衍生物的螢光探針:合成、光物理性質與形態學的研究

Developing 3-Hydroxyflavone Derivatives as Fluorescence Probes for MCF-7 Cells: Synthesis, Photophysical Properties and Morphology Studies

指導教授 : 陳昭岑

摘要


本論文的主要目的是合成一系列具有螢光團的雌二醇衍生物(estradiol derivatives)並研究其用於生物體內的雌性激素(Estrogen)受體之造影的可能性。 雌性激素在生長、發育、體內平衡,並且在調節細胞內重要的訊息傳遞途徑中具有舉足輕重的地位。雌性激素的傳統機制為藉由和雌性激素受體結合而活化雌性激素受體形成前起始複合體(preinitiation complex),使之進入細胞核和DNA特殊的調節序列:estrogen response element (ERE) 結合,進而影響生物體內蛋白質的生合成。在此設計及合成一系列的3-hydroxyflavone (3-HF) 的雌二醇螢光探針:HF-EDIOL、HF-7α-EDIOL、HF-EDINH2、HF-EDISO4H。其分子結構是在雌二醇上接一連接端,並在此連接端接上3-HF螢光團。選擇此螢光團的主要原因是因為它具有雙螢光放射峰,會隨著周圍環境極性的不同而造成螢光強度比例上(excited state intramolecular charge transfer/excited state intramolecular proton transfer ratio)的改變。希望能藉由此螢光特性,除了能偵測雌性激素受體位置之外,更能及時追蹤雌性激素的路徑。其中,HF-EDIOL和HF-7α-EDIOL的差異在於由雌二醇鄰接出的位置,分別是接在雌二醇的3號和7α的位置,想要探討雌性激素受體對此兩種化合物辨識的差異和在細胞中分佈的不同。另外亦有合成帶有正、負電荷的HF-EDINH2和HF-EDISO4H。由螢光光譜的探討可以發覺當螢光探針在DMSO和水的混合溶液中,隨著水濃度上的增加上述四個螢光探針都有聚集的現象產生,進一步利用DLS、zeta potential、SEM、TEM可以證實。在細胞實驗中,由雷射掃瞄共軛焦顯微鏡可發現HF-EDIOL主要分佈在細胞質中,在細胞核的量較少,HF-7α-EDIOL有較多進入細胞核中並且是以ESICT的放射光為主。

並列摘要


The aims of this thesis are to synthesize a series of estradiol-3-hydroxyflavone fluorescent probes and study the feasibility of using these probes for cellular trafficking of estradiol in MCF-7 cells. It was shown that estrogen plays an important role of growth, development and physiological balance. Furthermore, it has a vital character in the regulation of intracellular signaling pathway. This classical pathway of estrogen involves the formation of preinitiation complex (estrogen-ER complex) and the subsequent binding of this complex to estrogen response element (ERE) sequences of estrogen-responsive genes in nucleus, which leads to the production of associated proteins and finally a physiological response. Thus we design and synthesize a series of fluorescent probes by conjugation of 3-hydroxyflavone fluorophore with estradiol: HF-EDIOL, HF-7α-EDIOL, HF-EDINH2, and HF-EDISO4H. The fluorescent molecular probe features the estradiol derivative and a fluorophore, which is 3-hydroxyflavone, bridged by a hydrocarbon linker. The structural difference between HF-EDIOL and HF-7α-EDIOL is the linkage site, the derivatization at either 3 or 7α of the estradiol. The effect of positive and negative character in estradiol is probed by HF-EDINH2 and HF-EDISO4H. 3-Hydroxyflavone exhibits remarkable dual emission and possesses high sensitivity to different microenvironmental polarity in fluorescent wavelength of ESICT/ESIPT ratio change. Taking advantage of this unique photoproperties, the probe potentially can detect not only the location of estrogen receptor but provide direct tracking of its binding pathway in real time. In the studies of fluorescence spectra, as the ratio of DMSO and water solution increases, we obtained that fluorescence probes could self-assembly to certain kinds of aggregations. By utilizing DLS, zeta potential, TEM, and SEM technology, we can confirm the structure of these aggregations. In the cell imaging experiments, it reveals that HF-EDIOL can only be found in the cytoplasm while HF-7α-EDIOL distributed in both cytoplasm and nucleus with different ratios of ESICT and ESIPT emissions, displaying the location dependence.

參考文獻


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