本文選題：生物正交化學(xué)　切入點(diǎn)：BrdU　出處：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文　論文類型：學(xué)位論文

【摘要】：細(xì)胞增殖是生物體重要的生命特征,也是生物體生長(zhǎng)、發(fā)育、繁殖以及遺傳的基礎(chǔ)。細(xì)胞增殖檢測(cè)技術(shù)被廣泛地應(yīng)用于抗腫瘤藥物篩選、DNA復(fù)制的研究、干細(xì)胞研究、有機(jī)化合物和納米材料的毒性評(píng)價(jià)以及腫瘤放射敏感性評(píng)價(jià)等。檢測(cè)DNA復(fù)制的方法在單細(xì)胞水平直接檢測(cè)細(xì)胞增殖,是檢測(cè)細(xì)胞增殖最準(zhǔn)確的方法。BrdU/抗體免疫,EdU/點(diǎn)擊化學(xué),VdU/Inverse Diels-Alder反應(yīng)是當(dāng)前最常見的三種檢測(cè)DNA復(fù)制的方法。這些檢測(cè)細(xì)胞增殖的方法都有各自的優(yōu)缺點(diǎn),其中的一些不足會(huì)限制它們的應(yīng)用。因此,發(fā)展一種新的方法可以彌補(bǔ)這些方法的不足之處,為細(xì)胞增殖檢測(cè)提供更多的選擇。Suzuki-Miyaura反應(yīng)是有機(jī)硼化合物與有機(jī)鹵或者有機(jī)擬鹵在鈀或者鎳的催化下發(fā)生的交叉偶聯(lián)反應(yīng),廣泛應(yīng)用于有機(jī)合成。近些年該反應(yīng)也被用于修飾及標(biāo)記多糖、蛋白質(zhì)、多肽及寡核苷酸和DNA等細(xì)胞組份。我們希望將Suzuki-Miyaura化學(xué)偶聯(lián)方法應(yīng)用于標(biāo)記增殖細(xì)胞DNA中所摻入的BrdU,從而實(shí)現(xiàn)細(xì)胞增殖的檢測(cè)。我們首先在70℃的條件下以BrdU和苯硼酸為底物在四種溶劑中驗(yàn)證了該反應(yīng)的可行性。然后,在37℃使用不同的催化體系優(yōu)化反應(yīng)。結(jié)果表明使用n-Bu4N+OH-為堿時(shí)得到最佳實(shí)驗(yàn)結(jié)果,可以進(jìn)一步應(yīng)用于增殖細(xì)胞標(biāo)記。在合成七種含硼酸基的染料的基礎(chǔ)上,經(jīng)過化學(xué)反應(yīng),DNA分子水平以及細(xì)胞水平的多次實(shí)驗(yàn)摸索,以排除各種不利于細(xì)胞Suzuki-Miyaura偶聯(lián)標(biāo)記的因素。排除這些因素后,初步獲得了可以用于增殖細(xì)胞標(biāo)記的條件。經(jīng)重新優(yōu)化BrdU和苯硼酸的反應(yīng)并獲得最佳反應(yīng)條件后,使用三種染料硼酸和BrdU為模板的反應(yīng)表明染料硼酸可以和BrdU生成相應(yīng)的偶聯(lián)產(chǎn)物。在固定化細(xì)胞中,考察在四種條件下PdNP催化偶聯(lián)標(biāo)記,結(jié)果發(fā)現(xiàn)氫氣保護(hù)條件下可得到滿意的結(jié)果。該方法下,DTBPPS支持的鈀納米顆粒和摻入細(xì)胞中的BrdU在氬氣保護(hù)下發(fā)生氧化加成,再在氫氣條件下與染料硼酸完成偶聯(lián)標(biāo)記。進(jìn)一步優(yōu)化染料濃度,鈀的用量以及標(biāo)記時(shí)間,從而得到最佳增殖細(xì)胞標(biāo)記條件。我們使用不同方法驗(yàn)證了 BrdU/Suzuki-Miyaura偶聯(lián)標(biāo)記方法的可靠性。結(jié)果顯示,1)HepG2,SH-SY5Y,MDA-MB-231,HeLa以及A172細(xì)胞中遞增BrdU濃度和偶聯(lián)標(biāo)記的熒光強(qiáng)度成正比;2)HepG2細(xì)胞中遞增BrdU孵育時(shí)間標(biāo)記的熒光強(qiáng)度成正比;3)用Aphidicolin阻滯細(xì)胞周期可阻斷標(biāo)記;4)BrdU-EdU共定位實(shí)驗(yàn)表明BrdU/Suzuki-Miyaura偶聯(lián)標(biāo)記與EdU/點(diǎn)擊化學(xué)標(biāo)記兩種方法互相兼容,且兩種方法都特異性的標(biāo)記增殖細(xì)胞。將該方法應(yīng)用于腫瘤組織中檢測(cè)增殖細(xì)胞,結(jié)果表明經(jīng)過偶聯(lián)標(biāo)記后,可以很容易地區(qū)分增殖細(xì)胞和未增殖細(xì)胞。與EdU/點(diǎn)擊化學(xué)的方法聯(lián)合以用于追蹤DNA合成的Pulse-Chase實(shí)驗(yàn),結(jié)果表明增殖細(xì)胞被單色或兩色標(biāo)記,兩種標(biāo)記信號(hào)在時(shí)間和空間上具有可辨性。使用綠色熒光染料硼酸DEAC-硼酸及FITC-硼酸標(biāo)記增殖細(xì)胞,結(jié)果表明兩種硼酸染料均能特異性地標(biāo)記擴(kuò)增細(xì)胞,DEAC硼酸標(biāo)記結(jié)果理想而FITC硼酸的熒光背景較高�？傊�,我們發(fā)展了一種利用BrdU的細(xì)胞內(nèi)Suzuki-Miyaura偶聯(lián)反應(yīng)檢測(cè)細(xì)胞增殖的方法,該方法具有特異性好、重復(fù)性高、便宜、與EdU兼容等優(yōu)點(diǎn),具有良好的應(yīng)用價(jià)值。
[Abstract]:Cell proliferation is an important feature of biological life, but also the organism growth, development, reproduction and genetic basis. Cell proliferation detection technology is widely used in the screening of anti-tumor drugs, the study of DNA replication, stem cell research, evaluation of toxic organic compounds and nano materials and tumor radiosensitivity evaluation method for detection of DNA replication. At the single cell level direct detection of cell proliferation,.BrdU/ antibody detection method is the most accurate cell proliferation EdU/ VdU/Inverse Diels-Alder, click chemistry reaction is three. The most common method of DNA replication. These methods detect cell proliferation has its own advantages and disadvantages, some of these shortcomings will restrict their application development deficiencies. Therefore, a new method can make up for these methods, provide more choice for.Suzuki-Miyaura cell proliferation assay Organic boron compounds and organic halogen or halogen organic quasi cross coupling reactions in the catalytic palladium or nickel occurred, widely used in organic synthesis in recent years. The reaction was also used for modification and labeling of polysaccharides, proteins, peptides and oligonucleotides and DNA cell groups. We hope the chemical coupling method is applied to Suzuki-Miyaura the proliferation of DNA cells labeled by incorporation of BrdU, so as to realize the detection of cell proliferation. We first at the temperature of 70 DEG C to BrdU and phenylboronic acid as the substrate to verify the feasibility of the reaction in four solvents. Then the catalytic reaction system optimization of different at 37 degrees. The results show that the optimum the results of using n-Bu4N+OH- as the base, can be further applied to cell proliferation marker. Based on the synthesis of seven containing boronic acid dyes, through chemical reaction, DNA molecular level and cell level several times Experiments, in order to eliminate various factors not conducive to cell Suzuki-Miyaura coupling markers. Exclude these factors, we obtained can be used for cell proliferation marker conditions. After re optimizing BrdU and phenylboronic acid reaction and obtain the best reaction conditions, the use of three kinds of dye boron acid and BrdU as the template reaction showed that the dye and boric acid BrdU generates the corresponding coupled products. In immobilized cells, effects of marker PdNP catalyzed coupling in four conditions, the results indicate that the hydrogen protection under the condition of satisfactory results can be obtained. This method, DTBPPS supported palladium nanoparticles and the incorporation of BrdU in the cells of oxidative addition in argon atmosphere, and then in the condition of hydrogen and dye coupling markers. To further optimize the completion of boric acid dye concentration, dosage and time of palladium markers, in order to get the best cell proliferation marker conditions. We use different methods To verify the reliability of BrdU/Suzuki-Miyaura coupling marking method. The results showed that, 1) HepG2, SH-SY5Y, MDA-MB-231, fluorescence intensity is proportional to the increase of BrdU concentration and coupling marker HeLa and A172 cells; 2) the fluorescence intensity is proportional to the increase in HepG2 cells incubated with BrdU time marker; 3) using Aphidicolin to block the cell cycle block mark BrdU-EdU; 4) Co localization experiments show that the coupling of BrdU/Suzuki-Miyaura markers and EdU/ markers in two species of click chemistry method compatible with each other, to mark cell proliferation and two kinds of methods are specific. This method is applied to the detection of proliferating cells in tumor tissues, the results showed that after coupling after labeling can distinguish easily without cell proliferation and cell proliferation with the method of EdU/. Click chemistry with Pulse-Chase DNA for tracking experiment results show that the synthesis, proliferation of cells were labeled with monochromatic or white, two marker signal It can in time and space. Using green fluorescent dye DEAC- FITC- boric acid boric acid and boric acid labeled cells, the results showed that two boric acid dyes were specifically amplified cells, DEAC and FITC markers with the ideal borate boric acid high background fluorescence. In short, we developed a Suzuki-Miyaura coupling reaction by BrdU the cells within the cell proliferation assay, this method has good specificity, high repeatability, cheap, and compatible with the advantages of EdU, has good application value.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q2-33
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本文編號(hào)：1583600