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题名: DNA酶解级联微反应器与DNA氧化损伤分析研究
作者: 徐田
学位类别: 硕士
答辩日期: 2016-05
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 尹俊发 ; 汪海林
关键词: 级联酶微反应器,基因组DNA酶解,DNA氧化损伤,8-oxodG分析,纳米银 ; cascade microreactor, genomic DNA digestion, oxidative DNA damage, 8-oxodG analysis, Silver nanoparticles
其他题名: Cascade microreactor for enzymatic digestion of DNA and its application in detection of oxidative DNA damages
学位专业: 环境科学
中文摘要:       DNA氧化损伤与癌症、人体衰老及退行性疾病的发生密切相关。紫外光照射、环境污染物的暴露以及新型纳米材料的广泛应用都可能引起细胞内活性氧水平的升高进而引起DNA氧化损伤。8-oxodG作为一类重要的DNA氧化损伤标志物,在人体血样、尿样、细胞、组织等生物样品中广泛检测用于评价环境污染毒物的暴露与人类疾病发生的风险。
      高效液相色谱串联质谱(HPLC-MS/MS)因为灵敏度高、准确性好是检测DNA氧化损伤产物的“金标准”,但检测前需要通过脱氧核糖核酸酶(DNase I)、磷酸二酯酶(SVP)和碱性磷酸酶(ALPase)将基因组DNA酶解成单核苷产物。溶液体系下的酶解耗时长(>12h),酶解结束还需超滤除酶,氧气及溶液中痕量金属离子容易引起dG的氧化为8-oxodG的检测带来干扰。目前,酶反应器因为酶载量高,酶活性损失小,稳定性好在蛋白组学已经有了非常成熟的应用。而酶反应器在基因组DNA的酶解和DNA加和物定量检测方面的研究尚属空白,因  此本文提出发展一种可将基因组DNA酶解为单核苷的DNase I-SVP-ALPase级联酶反应器。
      本研究采用溶胶-凝胶法制备毛细管硅胶整体柱,通过戊二醛法将酶固载于整体柱制备DNase I,SVP及ALPase酶反应器。三种单酶反应器依次串联构建DNase I-SVP-ALPase级联酶反应器。实验对酶反应器进行了电子显微镜表征,荧光显微镜表征,酶载量和酶活检测。结果表明酶反应器通透性好和背压低,三种酶均成功地键合到整体柱上并具有高的酶载量(1.25~1.71 mg/mL)。此外,高的酶密度使得酶反应器酶活要高于溶液体系。随后,我们对级联酶反应器酶解小牛胸腺DNA(ct-DNA)得到单核苷产物的酶切效率进行评价,结果显示酶反应器酶解效率(45 min时酶切效率达到99.3%)要远高于溶液酶解体系(6 h时酶切效率为93.0 %)。级联酶反应器用于酶解DNA的重复性和稳定性好,可以重复使用和长期保存。
       将级联酶反应器应用于苯醌化合物/H2O2体外诱导ct-DNA氧化损伤效应的研究。首先,建立了基于级联微酶反应器酶解离线串联LC-MS/MS定量分析8-oxodG的方法。然后,通过不同的苯醌化合物/H2O2体外诱导ct-DNA产生氧化损伤,DNA样品采用级联酶反应器酶解结合UHPLC-MS/MS定量分析 8-oxodG水平,实验结果与溶液体系下酶解的检测结果相近,证明所构建的级联酶反应器可用于基因组DNA的有效酶解和8-oxodG的准确定量。酶反应器体系比溶液酶解体系更具有优势:高的酶解效率有利于减小酶解过程带来的DNA氧化;省去了溶液体系酶解的除酶过程,样品可直接质谱进样分析,提高分析效率。
       纳米银作为杀菌剂在医疗领域中已有广泛的应用。肝脏是人体重要的代谢器官,为了研究不同尺度的纳米银对HepG2肝癌细胞的DNA氧化损伤效应,我们分别采用20,50,100 nm的纳米银暴露HepG2细胞,考察纳米银对HepG2细胞的DNA氧化损伤效应。结果表明,纳米银能够诱导细胞ROS和8-oxodG水平的升高,且诱导趋势为20 nm > 50 nm > 100 nm。我们还将构建的级联酶反应器应用于了纳米银对HepG2细胞氧化损伤效应研究,证明我们构建的级联酶反应器可以用于细胞DNA的酶解和8-oxodG的定量分析。
      本研究成功地构建了可将基因组DNA有效酶解为单核苷的级联酶反应器,结合HPLC-MS/MS可用于8-oxodG的准确定量。该级联酶反应器有望进一步应用于基因组DNA样品中DNA加和物的快速鉴定和高灵敏分析。
英文摘要:       Oxidative DNA damage has been supposed to be associated with the development of cancer, aging and degenerative diseases. The generation of reactive oxygen species (ROS) arising from exposure to UV irradiation, environmental pollutants (such as halogenated p-benzoquinones) and novel nanoparticles (such as TiO2 and AgNPs) may result in oxidative DNA damage.8-Oxo-7,8-dihydro-2’-deox--yguanosine (8-oxodG) as a biomarker of oxidative DNA damages has been intensively investigated in human tissues, blood and urine to evaluate the development risk of disease.
       Liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method is an ideal tool for screening of 8-oxodG in various bio-samples due to its highly sensitivity and accuracy. Prior to HPLC-MS/MS analysis, DNA samples need to be digested to produce nucleosides. The digestion process in solution system is time-consuming (> 12h) and may introduce unexpected contamination of 8-oxodG which is easily induced by O2 and trace metal ions. Recently, enzyme-immobilized bioreactors have been used in many applications including the chemical analysis of proteins, kinetic studies and biocatalysis. However, there has no attempt been made in developing a bioreactor for genomic DNA digestion and quantitative detection of DNA adducts. In this paper, a novel DNase I-SVP-ALPase cascade bioreactor that can digest genomic DNA samples to produce single nucleosides was constructed.
      The preparation of monolith column was performed by sol-gel method. Then, enzymes were immobilized onto the monoliths by using glutaraldehyde reagent. Finally, the cascade bioreactors were assembled by tandem connecting the enzyme-immobilized capillary microreactors in an order of DNase I-SVP-ALPase. The bioreactors were characterized by scanning electron microscopy (SEM), fluorescence microscope, immobilization capacity and enzymatic activity assay. The results have shown that bioreactors have good permeability and low back pressure. The enzymes were immoblized onto monoliths successfully and present high immobilization capacity (1.25~1.71 mg/mL). In addition, enzymatic activity of bioreactor was higher than that in solution because of high immobilization density. By application of cascade bioreactor, ct-DNA has been digested efficiently and calculated enzymatic efficiency can reach 99.3 % within 45min, which is much higher than that in solution digestion system (only 93.0 % within 6 h). The remarkable stability of the immobilized enzymes guarantees the reusability and longtime preservation of the cascade bioreactor.
      The 3-enzyme cascade bioreactor was applied for screening of 8-oxodG in ct-DNA induced by halogenated quinones and H2O2. Firstly, the quantitative method was established based on the cascade bioreactor offline coupled to LC-MS/MS. Then, ct-DNA induced by halogenated quinones / H2O2 was digested by cascade bioreactor for UHPLC-MS/MS analysis of 8-oxodG. The detection results were highly close to that in solution system, indicating bioreactor system was powerful for genomic digestion to release single nucleosides and 8-oxodG. What’s more, the excellent enzymatic hydrolysis velocity improve analysis efficiency and may greatly reduce artifactual interferes of 8-oxodG. The collected products could be directly injected into UHPLC-MS/MS without tedious ultrafiltration process, which make it possible to realize on-line analysis by coupling bioreactor to instruments.
        Silver nanoparticles (AgNPs) acted as bactericides have been widely used in medical applications. In this study, we investigated the oxidative DNA damages induced by AgNPs (20 nm, 50 nm, 100 nm) in HepG2 cells. The result showed that AgNPs could induce the raise of ROS and then further the increase of 8-oxodG level. Size-dependent effect of AgNPs towards HepG2 cells was found. The capability of AgNPs to induce DNA oxidative damage was found in a size order of: 20 nm > 50 nm > 100 nm. Attempt has also been made to apply the cascade bioreactor in the digestion of genomic DNA isolated from HepG2 cells exposured by AgNPs. The result suggested that the cascade bioreactor can also been used for quantitative analysis of 8-oxodG in the cultured cells.
       In summary, our work demonstrated that the cascade monolithic bioreactor was powerful for HPLC-MS/MS analysis of 8-oxodG and are promising in rapid identification and highly-sensitive detection of adducts in genomic DNA samples.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/37031
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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Recommended Citation:
徐田. DNA酶解级联微反应器与DNA氧化损伤分析研究[D]. 北京. 中国科学院研究生院. 2016.
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