本文選題：FRET型小分子熒光探針 + 設(shè)計(jì)與合成��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：細(xì)胞內(nèi)pH(intracellular pH,pH_i)和pH穩(wěn)態(tài)對(duì)參與控制真核細(xì)胞的生長(zhǎng)和代謝極為重要。真菌細(xì)胞的pH_i影響真菌細(xì)胞膜的跨膜轉(zhuǎn)運(yùn)、代謝酶的活性和生物大分子的合成,同時(shí)也是一些其它一些細(xì)胞反應(yīng)的觸發(fā)器,如誘使熱敏蛋白的產(chǎn)生。一些具有抗真菌活性的藥物如質(zhì)子泵抑制劑奧美拉唑、抗心律失常藥胺碘酮的作用機(jī)理也與降低真菌細(xì)胞pH_i改變有關(guān)。準(zhǔn)確測(cè)定真菌細(xì)胞的pH_i有利于研究真菌細(xì)胞pH_i調(diào)控系統(tǒng),和pH_i相關(guān)的藥物作用機(jī)制。目前常用的測(cè)定真菌細(xì)胞內(nèi)pH的方法主要有pH敏感的小分子參比熒光探針和pH敏感的參比熒光蛋白。參比熒光蛋白雖然具有非侵襲性的優(yōu)點(diǎn),但操作復(fù)雜耗時(shí),在此不做深究。小分子熒光探針技術(shù)操作方便快捷,靈敏度高,但目前應(yīng)用于真菌細(xì)胞的小分子熒光探針較少,且大都為商品化的染料。小分子自身也存在一些局限性:1.不容易透過(guò)真菌的細(xì)胞膜,需要做成非熒光形式的二乙�；ヮ惙肿舆M(jìn)入細(xì)胞后再由酶水解得到熒光核,而且有些染料分子在細(xì)胞內(nèi)水解不完全,影響在細(xì)胞內(nèi)的成像;2.某些染料分子的pKa偏高,并不適合真菌細(xì)胞pH_i的成像和測(cè)定。本文的研究目的是設(shè)計(jì)合成新型的pH敏感的小分子熒光探針用于真菌細(xì)胞pH_i測(cè)定和成像。硼酯螺環(huán)可逆開(kāi)關(guān)控制的熒光團(tuán)R-B為本文課題組前期合成得到的pH敏感的小分子熒光探針,其結(jié)構(gòu)和機(jī)理新穎,但其pKa為9.46,并不適合真菌細(xì)胞生理學(xué)范圍內(nèi)的pH_i測(cè)定與成像。為了改善pKa和光譜性質(zhì),我們考慮將熒光團(tuán)R-B作衍生化處理,設(shè)計(jì)成一個(gè)基于FRET機(jī)理的參比型熒光探針。即將pH敏感的硼酯螺環(huán)母體結(jié)構(gòu)作為能量受體分子,香豆素?zé)晒夂藶槟芰抗w分子,剛性的哌啶環(huán)為中間連接體,設(shè)計(jì)合成得到了基于FRET機(jī)理的第三代pH敏感的小分子熒光探針R-BC。通過(guò)光譜性質(zhì)測(cè)定,我們得到R-BC的pKa為6.25,比較符合測(cè)定真菌細(xì)胞pH_i及酸化的pKa要求。同時(shí)該探針對(duì)H+的傳感具有良好的可逆性和專一性。通過(guò)探針應(yīng)用于真菌細(xì)胞,R-BC細(xì)胞膜通透性高、細(xì)胞毒性小,具有良好的真菌細(xì)胞生物相容性。經(jīng)過(guò)真菌細(xì)胞原位校正,R-BC的兩波段的熒光強(qiáng)度比Ired/Iblue在pH_i為5.0到8.0范圍內(nèi)與pH_i成良好的線性相關(guān),能準(zhǔn)確測(cè)定此范圍內(nèi)的pH_i。我們利用該探針測(cè)定了抗心率失常藥胺碘酮對(duì)于受試白色念珠菌的pH_i的影響,發(fā)現(xiàn)胺碘酮同樣能引起白色念珠菌的pH_i酸化,證明了胺碘酮發(fā)揮抗菌活性的部分作用機(jī)制與影響真菌細(xì)胞的pH_i有關(guān)。同時(shí)我們?cè)O(shè)計(jì)合成了哌啶端分別連有乙�；拖愣顾匾阴；呐瘐ヂ莪h(huán)探針?lè)肿覴-BN-1和R-BC-1。通過(guò)光譜性質(zhì)測(cè)定和比較其pKa,初步研究和探討了結(jié)構(gòu)修飾對(duì)硼酯螺環(huán)熒光核pKa的影響。結(jié)果表明接入的香豆素?zé)晒夂四艽偈够衔锓肿拥膒Ka向酸性區(qū)域移動(dòng),中間增加一個(gè)亞甲基連接單位能使pKa繼續(xù)向酸性區(qū)域移動(dòng),但由于兩熒光團(tuán)間隔距離增加及連接體剛性程度減弱,相應(yīng)的FRET效能降低�；贔RET機(jī)理的小分子熒光探針目前已經(jīng)廣泛應(yīng)用于哺乳動(dòng)物細(xì)胞,但還未有報(bào)道涉及到真菌細(xì)胞尤其是臨床上致病真菌的應(yīng)用。本文設(shè)計(jì)合成了新穎的FRET型的小分子熒光探針R-BC并將其應(yīng)用到了真菌細(xì)胞內(nèi)pH_i的測(cè)定與成像。該探針相對(duì)于商品化的熒光探針細(xì)胞生物相容性好,易透過(guò)真菌細(xì)胞膜,準(zhǔn)確測(cè)定的pH_i的范圍廣,為真菌細(xì)胞pH_i調(diào)控系統(tǒng)以及相關(guān)機(jī)理的研究提供了更有力的手段。
[Abstract]:The intracellular pH (intracellular pH, pH_i) and pH homeostasis are very important for controlling the growth and metabolism of eukaryotic cells. The pH_i of fungal cells affects the transmembrane transport of the fungal cell membrane, the activity of metabolic enzymes and the synthesis of biological macromolecules. It is also a trigger for some other cell counter responses, such as inducing the production of thermosensitive proteins. Antiarrhythmic drugs such as omeprazole, amiodarone, antiarrhythmic drug amiodarone, are also related to the reduction of pH_i changes in fungal cells. The accurate determination of pH_i of fungal cells is beneficial to the study of the pH_i regulatory system of fungal cells and the mechanism of drug action related to pH_i. Currently, it is commonly used for the determination of pH in fungal cells. The methods are mainly pH sensitive small molecular reference fluorescence probe and pH sensitive reference fluorescent protein. Although the reference fluorescence protein has the advantages of non invasive, it is complicated and time-consuming and does not study here. Small molecular fluorescence probe is convenient and fast and sensitive, but the small molecular fluorescent probes used in fungal cells are less, And most of them are commercialized dyes. Small molecules themselves also have some limitations: 1. it is not easy to penetrate the cell membrane of fungi and need to make a non fluorescent form of two acetyl ester molecules into the cell and then hydrolyze the fluorescent nucleus by enzymatic hydrolysis, and some dye molecules are not completely hydrolyzed within the cells, affecting the imaging in the cells; 2. some dyes are stained. The high pKa of the material molecules is not suitable for imaging and determination of pH_i in fungal cells. The purpose of this study is to design and synthesize a new pH sensitive small molecular fluorescent probe for the determination and imaging of pH_i in fungal cells. The fluorescent cluster R-B controlled by the reversible switch of borosate rings is a pH sensitive small molecule fluorophore synthesized in the earlier period of this group. The structure and mechanism of the needle are novel, but its pKa is 9.46, which is not suitable for pH_i determination and imaging in the scope of fungal cell physiology. In order to improve the pKa and spectral properties, we consider the fluorescence R-B as a derivative based fluorescence probe based on the FRET mechanism. The structure of the pH sensitive borate parent body is used as the energy. The receptor molecule, the coumarin fluorescent nucleus is an energy donor, and the rigid piperidine ring is the intermediate junction. The design and synthesis of the third generation pH sensitive small molecular fluorescent probe based on the FRET mechanism is determined by the spectral properties. We have obtained the pKa of R-BC of 6.25, which is in accordance with the pKa requirements for the determination of pH_i and acidification of fungal cells. The sensing of H+ has good reversibility and specificity. Through the application of the probe to fungal cells, the membrane permeability of R-BC cells is high, the cell toxicity is small, and the biocompatibility of the fungal cells is good. The fluorescence intensity in the two band of R-BC is better than that of the pH_i in the range of 5 to 8 pH_i in the range of pH_i in the range of 5 to 8. Sexual correlation, we can accurately determine the pH_i. in this range. We used the probe to determine the effect of amiodarone on the pH_i of Candida albicans. It is found that amiodarone can also cause pH_i acidification of Candida albicans. It is proved that the partial mechanism of amiodarone's antibacterial activity is related to the pH_i affecting the fungal cells. At the same time, we designed and synthesized the borate probe molecule R-BN-1 and R-BC-1. of piperidine and coumarin acetyl, respectively, to determine and compare their pKa through spectral properties. The effect of structural modification on the pKa of borate ring fluorescence nucleus was preliminarily studied and discussed. The pKa moves to the acidic region, and a methylene connection can be added to the pKa to move to the acid region, but the corresponding FRET efficiency is reduced due to the increase of the two fluorescent mass interval and the weakening of the rigidity of the connecting body. The small molecular fluorescent probe based on the FRET mechanism has been widely used in mammalian cells, but not yet. The report involves the application of fungal cells, especially the clinical pathogenic fungi. This paper has designed and synthesized a novel FRET type small molecular fluorescent probe R-BC and applied it to the determination and imaging of pH_i in the fungal cells. The probe is biocompatible with the commercialized fluorescent probe cells, and is easily permeable through the fungal cell membrane, and the accurate determination of P The wide range of H_i provides a powerful tool for the study of fungal cell pH_i regulation system and related mechanisms.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R914

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1 李冉;基于FRET機(jī)理的pH熒光探針的設(shè)計(jì)合成及真菌細(xì)胞pH_i成像[D];第二軍醫(yī)大學(xué);2017年

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本文編號(hào)：1857277