无需PCR扩增灵敏检测端粒酶活性新方法的研究

【作者】 高艳芳；

【导师】 金燕；

【作者基本信息】 陕西师范大学 ， 分析化学， 2016， 硕士

【摘要】 端粒(telomere)是存在于真核细胞线状染色体末端的一小段DNA-蛋白质复合体,它能保持染色体的完整性和控制细胞分裂周期。由于DNA的不完全复制机制,细胞每次分裂,端粒就会缩短一点,一旦端粒消耗殆尽,细胞凋亡机制就有可能被激活,细胞趋向凋亡。端粒酶(telomerase)是一种核蛋白逆转录酶,能在端粒3’末端催化合成端粒重复序列,使端粒不会由于细胞分裂而缩短,导致细胞永生化,成为肿瘤细胞。目前,已经在85%以上的肿瘤细胞中发现端粒酶呈高活性表达。因此,端粒酶被认为是一种普遍存在的肿瘤标示物。端粒酶活性的检测对于以端粒酶为靶点的肿瘤诊断和治疗至关重要。目前已建立多种端粒酶活性检测的方法,但仍存在灵敏度和可靠性等问题。因此,迫切需要发展能够简单快速、灵敏、可靠的检测端粒酶活性的方法。基于此,本文提出了端粒酶活性检测的新策略,旨在发展能够简单、快速、可靠、低成本的检测端粒酶活性灵敏的方法。本文的主要研究内容可总结为以下三方面：一、基于三重放大技术在单细胞水平均相检测端粒酶活性的荧光法基于三重放大技术,构建了一种无需PCR扩增、无须标记、灵敏检测端粒酶活性的荧光新方法。以G-四链体选择性染料,N-甲基卟啉二丙酸Ⅸ (NMM)为信号探针。以两个茎部隐藏有能形成G-四链体序列的发夹型核酸片段作为链替代反应(SDR)的DNA探针。端粒酶底物链在端粒酶的作用下,能够将短的底物链延长成一条含有多个TTAGGG重复序列的长DNA单链。此DNA延长单链能够与多个辅助DNA杂交,释放出许多引发DNA (T-DNA)去引发链替代反应,实现第一步信号放大。其次,链替代反应能够在两个打开的发夹末端形成两个G-四链体实现第二步信号放大。最后,重新释放的T-DNA能够引发下一轮的链替代反应,使NMM的荧光信号进一步累积增强。通过三重放大技术,在均相溶液中,可在1-3000个HeLa细胞提取物范围内灵敏地检测端粒酶的活性。同时,该方法能够检测正常细胞和不同种类肿瘤细胞中端粒酶活性表达的差异。此外,该方法也能用于评估端粒酶抑制剂对端粒酶活性的抑制效率。因此,该方法提供了一种简单、可靠、不需要荧光标记和酶辅助放大就能实现在单细胞水平灵敏检测端粒酶活性的新方法。二、基于金纳米颗粒信号放大灵敏检测端粒酶活性的荧光偏振法荧光偏振是一种可在均相溶液中灵敏、可靠、稳定地研究物质间相互作用的方法。为了实现简便可靠地检测端粒酶的活性,我们首次构建了无需PCR扩增,基于金纳米颗粒信号放大作用,在均相溶液中检测端粒酶活性的荧光偏振法。首先,巯基化的端粒酶底物链TS,通过金-硫键作用,自组装到金纳米颗粒表面。当存在端粒酶时,在底物链TS的3’末端延长出包含有多个TTAGGG重复序列的单链。这条延长链能够与许多条标记有羧基荧光素(FAM)的信号DNA分子(F-cDNA)互补,将一部分自由活动的F-cDNA捕获到金纳米颗粒表面。在金纳米颗粒的信号放大和端粒酶延长产物自放大的作用下,使F-cDNA的荧光偏振值FP增大。因此,在均相溶液中,在1-1000个HeLa细胞范围内能够实现端粒酶活性的快速检测。同时,利用此方法可以测定不同种类细胞中端粒酶活性的表达水平。此外,还可用该方法考察端粒酶抑制剂对端粒酶活性的抑制效率,这对于筛选以端粒酶为靶点的抗癌药物有潜在的应用价值。因此,我们构建了一种能够在单细胞水平实现端粒酶活性快速灵敏检测的方法,该方法具有简单可靠、响应速度快、成本低、灵敏度高等特点。三、基于三重放大技术检测端粒酶活性的比色法基于三重放大技术增强G-四链体DNA酶催化作用,建立了一种无需标记在均相溶液中检测端粒酶活性的比色法。首先在活性端粒酶存在时,其底物的延长产物能够与辅助DNA进行竞争杂交反应,释放多个链替代反应的引发DNA(T-DNA)。T-DNA能够引发发夹DNA H1和H2的自组装链替代反应,在H1：H2的两个末端分别形成G-四链体。氯化血红素(hemin)存在时,会形成G-四链体-hemin DNA酶,其具有类辣根过氧化酶的催化作用,能够催化H2O2-TMB体系显色。通过溶液颜色的变化或者吸光度值的变化,可灵敏地检测端粒酶的活性。利用裸眼能明显的区分出相当于10个HeLa细胞中端粒酶的活性,用吸光度值可以灵敏地检测到相当于1个HeLa细胞中端粒酶的活性。吸光度的变化与细胞个数在1-5000个细胞范围内呈现良好的线性关系。本方法还可以测定不同种类细胞中端粒酶的活性,同时还可考察端粒酶抑制剂对端粒酶活性的抑制效率。该方法为简便、低成本检测端粒酶活性提供了一种新思路。更多还原

【Abstract】 Telomeres are essential nucleoprotein structures that define the terminal segments of linear chromosomes in eukaryotes, and telomeres are essential for chromosome integrality and cell division. Because of DNA incomplete replication process, chromosome 3’ends progressively shorten during consecutive cell divisions, when lack sufficient telomeric repeats, the mechanism of apoptosis was activated. Human telomerase is a ribonucleoprotein reverse transcriptase that catalyzes the addition of the telomeric repeats onto the 3’-end of the human telomere chromosomes keeping telomeres extent, leading cell immortalization. Thus telomerase was selected as a universal tumor marker. The sensitive detection of telomerase activity could be crucially important for early cancer diagnosis and understanding the pathogenesis of disease. To date, a number of approaches to detect telomerase activity have been developed, some disadvantage such as sensitivity, assurance still exist. It is urgently needed to develop a high sensitive, rapid, reliable, cost effective strategy for telomerase activity detection. On the context, this paper puts forward a new strategy for telomerase activity detection aimed at developing simple, rapid, reliable, low-cost method of sensitive detection of telomerase activity.The main research contents and results are summarized as the following three sections.1. Fluorescent Detection of Telomerase Activity at Single-cell Level Based on Triple Amplification in homogeneous solutionHerein, we developed a PCR-free and label-free fluorescent strategy for facile, reliable and highly sensitive assay of human telomerase activity from crude cancer cell extracts. A G-quadruplex-selective fluorescent dye, N-methyl mesoporphyrin IX (NMM), was utilized as signal probe. Two hairpin probes with hidden G-quadruplex strand in their stem were designed as assembly components of strand displacement reaction (SDR). In this strategy, one telomerase reaction product contains several hexamer repeats which can hybridize with numerous assistant DNA to release a lot of trigger DNA (T-DNA) of SDR for achieving first step amplification. Then, strand displacement reaction led to the formation of G-quadruplex at the both end of two hairpin DNA probes for realizing second step amplification. Finally, the re-released T-DNA initiated another cycle of SDR, resulting in a significant increase in the fluorescence intensity of NMM. By taking advantage of triple signal amplification, the telomerase activity in the HeLa extracts equivalent to 1-3000 cells was detected in homogeneous solution. Telomerase activities of different cell lines, including cancer cells and normal cell, were also successfully evaluated. Meanwhile, the inhibition effect of 3’-azido-3’-deoxythymidine (AZT) was also investigated. Therefore, it offers a simple and reliable method for detecting telomerase activity at single-cell level without complex pre-modification of probe and enzyme auxiliary signal amplification, which has the merits of simplicity, rapid response, low cost and high reliability.2. Fluorescence Polarization Detection of Telomerase Activity at Single Cell Level Based on the Enhancement of Gold Nanoparticles Fluorescence polarization (FP) is a reliable, sensitive, and robust assay approach for studying biological interaction in homogeneous solution. To realize facile and reliable detection of telomerase activity, herein, for the first time, we developed a PCR-free fluorescent polarization strategy for sensitive detection of human telomerase activity at single cell level in homogenous solution based on gold nanoparticle (GNP) enhancement. Firstly, thiolated telomerase substrate (TS) primer is self-assembled onto the surface of GNP. In the presence of telomerase, TS primer was elongated to form a long single-stranded DNA containing numerous hexamer repeats (GGGTTA), which can hybridize with several short carboxyfluorescein (FAM)-modified complementary DNA (F-cDNA), leading to an increase in FP value due to the GNP enhancement and self-amplification of telomerase. So, the telomerase activity in the HeLa extracts equivalent to 1-1000 cells can be rapidly detected in homogeneous solution. Telomerase activities of different cell lines were also successfully evaluated. Meanwhile, the inhibition efficiency of telomerase inhibitor was studied, which holds great potential in screening telomerase-targeted anticancer drugs as well. Therefore, it offers a facile and reliable method for detecting telomerase activity at single-cell level, which has the merits of simplicity, rapid response, low cost, high sensitivity and reliability.3. Label-free Colorimetric Detection of Telomerase Activity via Triple AmplificationA label-free method is developed for colorimetry detecting telomerase activity using triple amplification mediated G-quadruplex-hemin DNAzymes. In this strategy, one telomerase reaction product contains several hexamer repeats which can hybridize with numerous assistant DNA to release a lot of trigger DNA (T-DNA). T-DNA can initiate cyclic strand displacement reaction between hairpin DNA H1 and H2. Through triple amplification, the simultaneous formation of two G-quadruplex at the both end of H1:H2 complex was occurred, the hemin-G-quadruplex DNAzyme can catalyze the H3O2-mediated oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB) to produce a colored product. Colorimetric functionality allows rapid preliminary discrimination of telomerase activity by the naked eye. Then the employment of UV-vis technique results in greatly improved detection sensitivity. Telomerase activity of 10 HeLa cells or above can be discriminated with the colorimetric functionality and UV-vis technique ensures that telomerase activity down to 1 HeLa cell can be sdetected. Telomerase-activities of different cell lines and telomerase inhibitor were also successfully investigated. With the ability of fast detection, outstanding sensitivity, and excellent selectivity, this strategy offers a convenient and specific method for telomerase activity detection, which exhibits great potential in the practical application in telomerase-based early stage cancer diagnosis.更多还原