類固醇螢光探針與金奈米粒子應用在標定及純化其相對結合蛋白的研究

Application of Steroid Derivatized Fluorescent Probes and Gold Nanoparticles to Label and Purify Corresponding Binding Proteins

梁明仁(Ming-Jen Liang)

指導教授 ： 陳昭岑

國立臺灣大學/理學院/化學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.02584

摘要

本論文的目的在於合成具有螢光團的類固醇衍生物，用於標定或偵測生物體內的類固醇結合蛋白及雌性素接受體，並且更進一步的把類固醇衍生物修飾在金奈米粒子上，用於分離純化此類固醇結合蛋白。
孕烯醇酮（pregnenolone）可以在斑馬魚胚胎中幫助穩定微管（microtubules）的結構進而影響細胞的移動。因此推測在斑馬魚胚胎中，可能具有孕烷醇酮的結合蛋白，而此結合蛋白可以調控微管的形成。在此我們設計並合成螢光探針來證實此推論，分子探針是在孕烯醇酮上接一連接端，並在此連接端末端接上 fluorescein。當此分子滲透至斑馬魚胚胎裡，再利用雷射掃瞄共軛焦顯微鏡（confocal microscope），觀看在斑馬魚胚胎裡孕烯醇酮結合的位置。同時我們也合成孕烯醇酮的連接端末端具有 3-Hydroxyflavone衍生物。此螢光團的選擇是因為它會因周圍環境極性的不同，而造成螢光波長或量子產率的改變，希望可藉此化合物偵測斑馬魚胚胎裡孕烯醇酮結合蛋白的位置。再者，我們亦使用金奈米粒子當固相載體，用於純化分離此結合蛋白，於是合成了末端支鏈有硫醇的孕烯醇酮衍生物，可將此分子固定在金奈米粒子上，藉此用來分離斑馬魚胚胎裡的細胞液中和孕烯醇酮有相互作用力的蛋白質。同時我們利用電泳和基質輔助雷射脫附游離-飛行時間質譜法（MALDI-TOF）來判斷是否為此蛋白質。我們也合成雌二醇（estradiol）衍生物用來進行對細胞內雌激素受體（estrogen receptor）做研究。而此分子探針是在雌二醇上接上一連接端，並在此連接端末端接上fluorescein，當此分子滲透細胞膜到細胞質時，希望可以和雌激素受體結合，藉此可直接在細胞中標定雌激素受體的位置，由雷射掃瞄共軛焦顯微鏡可發現此分子能滲透到細胞質中，並且由西方墨點法（Western blot analysis）證實雌激素二醇衍生物可以和雌激素受體結合，並且活化此受體，使之進入細胞核中。

關鍵字

螢光探針 ； 金奈米粒子 ； 孕烷醇酮

並列摘要

The aims of this thesis are to synthesize fluorescent steroid derivatives, which can be used to label the corresponding steroid binding protein and estrogen receptor in vivo, and to fabricate pregnenolone-capped gold nanoparticles for further purifying the pregnenolone binding protein.
It was shown that pregnenolone can help stabilize the structure of microtubules in zebrafish embryo and affects the cell movement. Presumably there might be pregnenolone binding protein, which can control the formation of microtubules in zebrafish embryo. Motivated by this observation and presumption, we designed and synthesized fluorescent probes to locate the corresponding binding protein. The fluorescent molecular probe features the pregnenolone derivative and a fluorophore, which can be fluorescein or 3-hydroxyflavone, bridged by a hydrocarbon linker. The result showed that the molecular probe incorporated with fluorescein can penetrate into the zebrafish embryo. By utilizing confocal microscope, the pregnenolone binding protein can be located with the aid of fluorescein antibody. The fluorescent probe, which consists of 3-hydroxyflavone derivatives, was also synthesized intending to serve different functions. 3-Hydroxyflavone is very sensitive to the microenvironmental polarity resulting in fluorescent wavelength or quantum yield change. Taking advantage of this unique photoproperties, the probe potentially can detect not only the location of pregnenolone binding protein but monitor its concentration variation in real time. The gold nanoparticles capped with pregnenolone derivative were also fabricated. These modified gold nanoparticles present multi cognate substrates for interacting with the pregnenolone binding protein, rendering the isolation of this protein from the crashed embryos. In combination of MALDI-TOF and polyacrylamide gel electrophoresis, the identity of the isolated protein will be verified.
The estradiol-fluorescein fluorescent molecular probe was also synthesized and applied to study the cellular trafficking of the estradiol. Western blot analysis revealed that the molecular probe indeed can penetrate into cells and bind with its receptor in the cytoplasm. Moreover, the ternary complex can further enter into the nucleus.

並列關鍵字

fluorescent probe ； gold nanoparticle ； pregnenolone

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