【作者】 刘斌；

【导师】 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2007， 博士

【摘要】 现代科学认为:许多疾病的发生与细胞内的基因结构和表达水平变化有着直接的联系。但是,如何借助有效的研究手段,从分子水平上实时、灵敏、特异地获取这些信息变化,尤其是基因表达信息的变化,已经成为医学研究所面临的严峻挑战,同时也为分析化学加强与医学的融合提供了新的机遇。荧光分子探针技术作为一种灵敏、准确的研究手段和方法,结合生物大分子的一些特殊生化性质,如核酸杂交、核酸-蛋白质的相互作用、蛋白质(酶)对底物的特异性识别和切割等,将生物分子信息转变为易于检测的荧光信号,有望在获取基因表达产物信息方面取得突破。但是,由于受到荧光分子探针类型的限制和基因表达产物多样性的影响,目前基于荧光分子探针检测基因表达产物的方法还不多。因此,进一步发展可用于检测基因表达产物的荧光分子探针新方法、新技术和新原理是一项非常有意义的前瞻性研究工作。本论文以细胞中能降低肿瘤发生风险的一类基因表达产物损伤修复蛋白为研究对象,利用这些蛋白的催化功能,结合研制新型荧光分子探针,从检测尿嘧啶糖苷酶(UDG)着手,以发展操作简单快速、灵敏度高、选择性好、而且样品量小的新方法为研究主线,利用新的双链荧光探针建立了损伤修复蛋白UDG、APE1和T4DNA连接酶的活性分析新方法,利用已有的分子信标探针开展了损伤修复蛋白hOGG1活性分析;同时利用分子信标开展了基因表达产物mRNA的定量检测研究。这种用于基因表达产物研究的荧光分子探针技术具有操作简单、快速、可实时监测等优点,一定程度上克服了基于放射性标记、凝胶电泳等经典分析方法费时、费力以及无法实时获取基因表达产物信息的缺陷。论文主要内容归纳如下:第一部分荧光分子探针用于基因表达产物碱基损伤修复蛋白的检测1、利用双链荧光探针建立了简单快速的UDG酶活性分析的新方法。UDG酶是一种能特异消除损伤碱基尿嘧啶的蛋白,在防止基因突变,调节免疫功能等方面具有重要意义。目前采用的放射性标记和电泳分析UDG酶活性的方法,不利于酶促反应过程实时监测和活性分析。本章设计双链荧光探针作为切割底物和检测分子,通过监测尿嘧啶切割过程中溶液荧光信号的变化,建立了UDG酶高灵敏分析方法,检测下限可达0.033U/mL;考察了反应溶液中金属离子、化学药物以及底物浓度等条件变化对切割反应初速度的影响,开展了UDG酶促反应机制和动力学过程研究;这种新方法还可快速、准确检测肿瘤细胞中UDG酶活性,检测灵敏度与放射性同位素法相当。以上结果不仅证明荧光分子探针技术用于碱基损伤修复蛋白活性检测的可行性,而且有望为临床诊断提供新的辅助手段。2、利用双链荧光探针建立了简单、快速的APE1酶活性分析的新方法。APE1是细胞中水解AP位点的糖苷酶,主要参与碱基损伤修复、调节细胞周期、细胞凋亡等生命过程。为了避免传统的APE1酶活性分析方法操作复杂、费时的缺陷,我们以含有AP位点的双链荧光探针作为反应底物,在均相溶液中实时监测AP位点水解反应过程,建立了一种APE1酶活性分析的新方法,其线性检测范围为0.024-2U/mL,检测下限为0.024U/mL;通过考察和优化溶液的金属离子、化学药物以及底物浓度等实验条件,在均相溶液中实现了酶促反应机制和动力学过程研究;这种新方法还可用于肿瘤细胞中APE1活性水平的检测,与其他方法相比,该方法不仅灵敏度达到放射性同位素法的水平,而且特异性强、检测结果准确,有望在肿瘤早期诊断上发挥重要作用。3、利用双链荧光探针建立了DNA连接酶活性分析的新方法。核酸连接是生命科学中倍受关注的生命活动。其活性分析和机理研究有助于进一步拓展核酸连接反应在疾病检测、基因载体及核酸修饰与分析等方面的应用。利用双链分子探针作为核酸连接的模板和信号分子,通过实时监测荧光信号降低来监测T4DNA连接酶反应过程,为连接酶活性分析、连接机理及其动力学过程研究提供了一种简便快捷的方法。4、利用单链探针分子信标建立了简单、快速的hOGG1酶活性分析的新方法。根据hOGG1酶能切割损伤碱基8-oxoG和裂解DNA链双重功能的特性,将8-oxoG设计在分子信标茎部,发展了一种可作为hOGG1酶底物的新型荧光探针。通过实时监测hOGG1酶识别切割8-oxoG后引起的溶液荧光信号变化,建立了一种分析hOGG1酶活性的新方法,检测线性范围为0.0125-5U/mL,检测下限为0.0125U/mL;通过考察和优化反应溶液中金属离子、化学药物以等实验条件,开展了hOGG1酶促反应机制和动力学过程研究;这种新的荧光分析方法用于多种肿瘤细胞hOGG1活性水平准确检测的结果表明:这种新方法可望为临床肿瘤早期诊断提供辅助手段。第二部分荧光分子探针用于基因表达产物mRNA的检测5、利用分子信标体外定量检测肿瘤抑制基因ING1 mRNA的表达。基于肿瘤抑制基因ING1 mRNA的序列保守区设计合成分子信标,将其与体外转录的ING1 RNA杂交,得到了ING1分子信标/RNA杂交标准曲线;通过考察杂交缓冲溶液的离子强度、pH值以及其他核酸分子对分子信标与细胞mRNA杂交过程的影响和体系优化,在均相溶液中实现了ING1 mRNA表达水平的定量检测,并利用RT-PCR法验证了检测结果。与其他mRNA检测方法相比,该方法简单、快速,而且检测过程不经过扩增步骤,进一步保证了实验结果的准确,还能用于基因芯片来发展高通量的检测方法。6、利用单链探针分子信标定量检测p21 mRNA的表达。为了进一步拓展分子信标在mRNA检测中的应用,在前一章的工作基础上,利用分子信标检测了p21 mRNA的表达水平。基于p21 mRNA的保守序列设计了分子信标,将其与通过基因重组和体外转录得到的p21 RNA杂交,构建了p21分子信标/RNA杂交标准曲线;通过考察杂交缓冲溶液的离子类型、浓度、pH值以及溶液中的核酸分子对分子信标检测法的影响和体系优化,在均相溶液中定量检测了基因转染和5-氟尿嘧啶处理的MCF-7细胞,以及RNAi处理的CNE2细胞中p21 mRNA的表达水平。这种检测技术操作简单,无需对细胞mRNA进行转录和扩增,即可直接实现靶基因mRNA表达水平的检测。7、5-氟尿嘧啶(5-FU)诱导的HNE1细胞ING1 mRNA表达变化检测与生物学特性分析。利用分子信标检测了5-氟尿嘧啶处理基因转染前后鼻咽癌HNE1总RNA杂交后引起的溶液荧光信号变化,用这种荧光信号变化值直接表示ING1 mRNA表达水平的高低;利用细胞生长速度检测法、流式细胞术、蛋白质杂交法和MTT法等分析手段考察ING1 mRNA表达变化对细胞生理特征的影响。对基于不同分析方法得到的实验结果进行比较分析,发现ING1 mRNA表达水平能作为判断细胞生长速度、细胞周期分布、蛋白质表达水平和对抗肿瘤药物敏感性的一个有效指标。更多还原

【Abstract】 Modern science thought that the development of many diseases is directly correlated with the gene structure and expression changes. However, how to sensitively and specifically obtain these information in real time, especially obtain the change of gene expression products by utilizing effective tool, has become a great challenge for medical science and provide a new chance for the crosslink of analytical chemistry with medical science at the same time. The fluorescent molecular probe technology, a kind of sensitive and accurate research tool and method, which utilized the biochemical specificity, including nucleic acids hybridization, interaction of DNA-protein, specific recognization and digestion of enzyme to substrates et al, can transfer the information of bio-molecular into easily detectable fluorescent signal and is hopeful on the detection of gene expression products. However, the methods which can be applied to detect the gene expression are limited for the complex of products and lack of fluorescent probes. So, further develop new methods, new technology and new principles, which can be applied on detection of gene expression products, is a meaningful work of prospective research.The dissertation, which focused on the detection of gene expression products, was begun from the detection of uracil-DNA glycosylase (UDG) by using the enzymatic activity of these proteins and combing with the development of new kinds of probes. A series of fluorescent methods not only for detection of base damage repair protein including UDG, APE1, T4DNA ligase and hOGG1, but also for the quantitative detection of mRNA was developed. The simple, fast and real time new fluorescent probe technology for the detection of gene expression products, overcomes the drawbacks of traditional methods, which need the complicated step of isotope labeling, electrophoresis and autoradiography and is incapable of acquiring the dynamic data in real time. The main details are presented as following:Part 1 Detection of base damage repair protein based on fluorescent molecular probes1. A novel method for real time detection of UDG based on double-stranded fluorescent probesUDG is a kind of repair protein which is important for inhibition of gene mutation and immunological regulation by removing damage base of uracil. Here, we introduce a novel method for monitoring uracil removal in real-time. The double-stranded DNA probes containing uracil residues, which FRET can occur, are used to monitor this process in homogeneous solution. The method not only overcomes the drawbacks of discontinuity, time consuming and complication which inherited in traditional assays of radioisotope and electrophoresis methods, but also can be applied to determine the kinetic constants of UDG. The low limitation of UDG assay is 0.033U/mL, the KM and kcat are 0.11μM and 4s-1 separately. In addition, the method has been applied to explore enzymatic mechanism and the effect of metal ions, chemical drugs and concentration of substrates on UDG activity. Finally, UDG activities in tumor cells were quickly and accurately detected by applying this method in vitro. The work not only confirmes the feasibility of protein activity detection by fluorescent technology, but also provides a stable basement for making it as a common detection method.2. A novel method for real time detection of APE1 based on double-stranded fluorescent probesAs an important glycosylase of base damage repair, APE1 is associated with the processes of base repair, cell cycle regulation and cell apoptosis. In order to avoid the drawbacks of complication and time-consuming, which inherited in the traditional methods, a new method, which can detect APE1 activity rapidly, sensitively and precisely, was established by real-time monitoring hydrolysis of AP site in the homogenous solution. The linear-detection range is 0.024-2U/mL and the limit of detection is 0.024U/mL. Additionally, the enzymatic mechanism and kinetics were studied by investigating the influence of external factors including metal ions, chemical drugs and substrates on APE1 activity. The method was finally applied to detect APE1 activity of A549 and Tca8113 tumor cells in vitro.(3) Study the ligation mechanism of T4DNA ligase based on double-stranded fluorescent probes. DNA ligase is a vital enzyme in the repair, replication and recombination of nucleic acids. The ligation mechanism is usually assayed by electeophoresis and autoradiography, which is complex and time-consuming. Based on double-stranded probes, the ligation mechanism of T4 DNA ligase has been studied conveniently. The method can be applied to investigat the ligation mechanism of other DNA and RNA ligases.4. A novel simple and rapid method for real time detection of hOGG1 based on molecular beaconsEvidence showed that the difference of tumor susceptibility is often caused by the change of hOGG1 activity. A new method, which can rapidly, sensitively assay the hOGG1 activity, was established based on molecular beacons. The method has been applied not only for investigating the effect of external factors including metal ions, chemical drugs and substrate on hOGG1 activity but also for studying the enzymatic mechanism and kinetics. It was further applied to detect hOGG1 activity of MCF-7, A549, Tca8113 and HNE1 tumor cells. Comparing with methods of double-stranded probes, it avoided the step of preparing duplexes.Part 2. Detection of gene mRNA based on molecular beacons5. Quantitative detection of p33ING1 mRNA based on molecular beaconsMolecular beacons, which contain complimentary sequences with conserve region of ING1 mRNA, are applied to construct a standard curve of ING1MB/RNA after hybridizing with ING1 cRNA. With the help of standard curve, the effects of drug treatment, gene transfection and p53 RNAi on ING1 mRNA levels were quantitatively detected in homogenous solution after investigating the influence of metal ions, pH value and other nucleic acids on ING1 MB/mRNA hybridization. The results were further confirmed by RT-PCR. Compared with RT-PCR and other methods, the simple and time-saving method showed high accuracy and reproducibility by avoiding the amplification step.6. Quantitative detection of p21 mRNA based on molecular beacons (MBs)The p21 mRNA levels were detected based on molecular beacons in order to further broaden the application of molecular beacons detection technology. Molecular beacons, which can specifically hybridize with conserve sequences of p21 mRNA were designed and a p21MB/RNA hybridization standard curve was constructed according to the fluorescent change of p21MB caused by different concentrations of p21 cRNA. After investigating the influence of many factors including ions, pH and nucleic acids molecules on this method, the p21 mRNA levels of tumor cell with drug treatment, gene transfection and p53 RNAi were quantitatively detected with the help of standard curve. From the results, it can be found that the simple technology can directly detect target mRNA without further steps of transcription and amplification.7. Detection of ING1 mRNA and biological characteristic analysis of HNE1 cellsThe fluorescent changes of ING1 molecular beacons caused by mRNA of HNE1 cells with gene transfection or not were detected after treatment with 5-fluoruracil. The intensities of fluorescent change were used to stand for the ING1 mRNA levels. In addition, the effect of ING1 mRNA levels on cell biological characteristics was studied by combing with other analytical tools including cell growth detection, flow cytometry, western blotting and MTT assays. The results showed that ING1 mRNA levels can be acted as an indicator of cell growth speed, distribution of cell cycle, protein expression and cell sensitivity to 5-fluoruracil.更多还原