基于BrdU的Suzuki-Miyaura偶联在检测细胞增殖中的应用研究

发布时间：2018-03-08 10:51

  本文选题：生物正交化学　切入点：BrdU　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：细胞增殖是生物体重要的生命特征,也是生物体生长、发育、繁殖以及遗传的基础。细胞增殖检测技术被广泛地应用于抗肿瘤药物筛选、DNA复制的研究、干细胞研究、有机化合物和纳米材料的毒性评价以及肿瘤放射敏感性评价等。检测DNA复制的方法在单细胞水平直接检测细胞增殖,是检测细胞增殖最准确的方法。BrdU/抗体免疫,EdU/点击化学,VdU/Inverse Diels-Alder反应是当前最常见的三种检测DNA复制的方法。这些检测细胞增殖的方法都有各自的优缺点,其中的一些不足会限制它们的应用。因此,发展一种新的方法可以弥补这些方法的不足之处,为细胞增殖检测提供更多的选择。Suzuki-Miyaura反应是有机硼化合物与有机卤或者有机拟卤在钯或者镍的催化下发生的交叉偶联反应,广泛应用于有机合成。近些年该反应也被用于修饰及标记多糖、蛋白质、多肽及寡核苷酸和DNA等细胞组份。我们希望将Suzuki-Miyaura化学偶联方法应用于标记增殖细胞DNA中所掺入的BrdU,从而实现细胞增殖的检测。我们首先在70℃的条件下以BrdU和苯硼酸为底物在四种溶剂中验证了该反应的可行性。然后,在37℃使用不同的催化体系优化反应。结果表明使用n-Bu4N+OH-为碱时得到最佳实验结果,可以进一步应用于增殖细胞标记。在合成七种含硼酸基的染料的基础上,经过化学反应,DNA分子水平以及细胞水平的多次实验摸索,以排除各种不利于细胞Suzuki-Miyaura偶联标记的因素。排除这些因素后,初步获得了可以用于增殖细胞标记的条件。经重新优化BrdU和苯硼酸的反应并获得最佳反应条件后,使用三种染料硼酸和BrdU为模板的反应表明染料硼酸可以和BrdU生成相应的偶联产物。在固定化细胞中,考察在四种条件下PdNP催化偶联标记,结果发现氢气保护条件下可得到满意的结果。该方法下,DTBPPS支持的钯纳米颗粒和掺入细胞中的BrdU在氩气保护下发生氧化加成,再在氢气条件下与染料硼酸完成偶联标记。进一步优化染料浓度,钯的用量以及标记时间,从而得到最佳增殖细胞标记条件。我们使用不同方法验证了 BrdU/Suzuki-Miyaura偶联标记方法的可靠性。结果显示,1)HepG2,SH-SY5Y,MDA-MB-231,HeLa以及A172细胞中递增BrdU浓度和偶联标记的荧光强度成正比;2)HepG2细胞中递增BrdU孵育时间标记的荧光强度成正比;3)用Aphidicolin阻滞细胞周期可阻断标记;4)BrdU-EdU共定位实验表明BrdU/Suzuki-Miyaura偶联标记与EdU/点击化学标记两种方法互相兼容,且两种方法都特异性的标记增殖细胞。将该方法应用于肿瘤组织中检测增殖细胞,结果表明经过偶联标记后,可以很容易地区分增殖细胞和未增殖细胞。与EdU/点击化学的方法联合以用于追踪DNA合成的Pulse-Chase实验,结果表明增殖细胞被单色或两色标记,两种标记信号在时间和空间上具有可辨性。使用绿色荧光染料硼酸DEAC-硼酸及FITC-硼酸标记增殖细胞,结果表明两种硼酸染料均能特异性地标记扩增细胞,DEAC硼酸标记结果理想而FITC硼酸的荧光背景较高。总之,我们发展了一种利用BrdU的细胞内Suzuki-Miyaura偶联反应检测细胞增殖的方法,该方法具有特异性好、重复性高、便宜、与EdU兼容等优点,具有良好的应用价值。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q2-33
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本文编号：1583600