基于氧化石墨烯和阳离子共轭聚合物的端粒酶活性分析
发布时间：2018-08-12 18:03
【摘要】：端粒酶是由RNA和蛋白质组成的核糖核蛋白酶,具有反转录活性。端粒酶与端粒DNA结合后,会以自身RNA序列为模板,进行不断的复制,阻止端粒DNA在细胞分裂过程中造成的末端缩短。端粒酶在人的正常细胞中很难被检测到活性,它一般在肿瘤或癌细胞中被激活。肿瘤或癌细胞的强大活性与端粒酶被激活密切相关。通过检测端粒酶活性可以实现肿瘤或癌症的早期诊断,同时对开发以端粒酶为靶标分子的抗癌药物具有重要意义。本学位论文分别利用氧化石墨烯和阳离子共轭聚合物,结合恒温指数扩增和杂交链式反应两种信号放大技术,建立了系列端粒酶活性检测的方法。主要研究内容如下:方法一:基于磁性氧化石墨烯和恒温指数扩增反应高灵敏度检测端粒酶活性。端粒酶延伸产物是具有-(ggttag)n重复序列的DNA。设计一条与两个重复序列相匹配的特异性的DNA探针-(ctaacc)2使其与端粒酶延伸产物杂交,形成双链DNA。过量的DNA探针被磁性石墨烯吸附,通过磁分离去除。利用恒温指数扩增反应(IEXPAR)对端粒酶延伸产物捕捉的DNA探针进行快速的扩增。用与双链DNA特异性结合的SYBR Green I染料对扩增产物进行实时荧光检测。在最优条件下,可以检测到低至50个癌细胞中的端粒酶活性。实现了恒温扩增条件下端粒酶活性的高灵敏度检测。方法二:基于阳离子共轭聚合物荧光共振能量转移检测端粒酶活性。基于阳离子共轭聚合物(PFP)与荧光染料SYBR Green I(SG)之间的荧光共振能量转移,建立了一种简单快速的端粒酶活性检测方法。当端粒酶存在时,引物探针被延伸,生成具有-(ggttag)n重复序列的DNA。然后通过链霉亲合素与生物素的特异性作用将端粒酶延伸产物连接在磁性微球上。加入与端粒酶延伸产物匹配的探针-(ctaacc)2。端粒酶延伸产物形成双链结构之后加入SG。SG能够特异性的嵌入到DNA双链结构中。最后,加入PFP。PFP通过静电作用与双链DNA结合,与嵌入在双链结构中的SG发生荧光共振能量转移(FRET)。因此,根据FRET的效率可以实现对端粒酶活性的定量检测。该方法可以检测到30万个Hela细胞中提取的端粒酶活性。为了进一步提高方法的灵敏度,我们将该方法与杂交链式反应(HCR)结合,实现了检测信号的放大,灵敏度提高了一个数量级,可以检测到6万个Hela细胞中的端粒酶活性。此方法操作简单,检测成本低,但检测灵敏度还有待于进一步提高。
[Abstract]:Telomerase is a ribonucleoprotease composed of RNA and protein with reverse transcriptional activity. When telomerase binds to telomere DNA, it uses its own RNA sequence as a template for continuous replication to prevent telomere DNA from shortening the end during cell division. Telomerase activity is difficult to detect in human normal cells and is generally activated in tumors or cancer cells. The strong activity of tumor or cancer cells is closely related to the activation of telomerase. The detection of telomerase activity can realize the early diagnosis of tumor or cancer, and it is of great significance for the development of anticancer drugs targeting telomerase. In this dissertation, a series of telomerase activity detection methods were established by using graphene oxide and cationic conjugated polymers, combined with two signal amplification techniques: constant temperature index amplification and hybrid chain reaction. The main contents are as follows: method 1: telomerase activity was detected with high sensitivity based on magnetic graphene oxide and isothermal index amplification reaction. Telomerase extension products are DNAs with-(ggttag) n repeats. A specific DNA probe, (ctaacc) 2, which matched two repeat sequences, was designed to hybridize with telomerase extension products to form double-stranded DNAs. Excessive DNA probe was adsorbed by magnetic graphene and removed by magnetic separation. The DNA probe captured by telomerase extension product was rapidly amplified by isothermal index amplification reaction (IEXPAR). The amplified products were detected by real-time fluorescence with SYBR Green I dyes which were specifically bound to double-stranded DNA. Telomerase activity can be detected in as low as 50 cancer cells under optimal conditions. The high sensitivity detection of telomerase activity under constant temperature amplification was realized. Method two: telomerase activity was detected based on fluorescence resonance energy transfer of cationic conjugated polymer. Based on the fluorescence resonance energy transfer between cationic conjugated polymer (PFP) and fluorescent dye SYBR Green I (SG), a simple and rapid method for detecting telomerase activity was developed. In the presence of telomerase, the primer probe was extended to produce DNAs with-(ggttag) n repeats. The telomerase extension product was then connected to the magnetic microspheres by the specific interaction between streptavidin and biotin. A probe matching telomerase extension product, (ctaacc) 2, was added. Telomerase extension products can be specifically embedded into the double stranded structure of DNA after the addition of SG.SG after the formation of double strand structure. Finally, the addition of PFP.PFP binds to double-stranded DNA by electrostatic interaction, and generates fluorescence resonance energy transfer (FRET). With SG embedded in double-stranded structure. Therefore, the quantitative detection of telomerase activity can be realized according to the efficiency of FRET. The method can detect telomerase activity extracted from 300, 000 Hela cells. In order to further improve the sensitivity of the method, we combine the method with hybrid chain reaction (HCR) to amplify the detection signal. The sensitivity is increased by an order of magnitude, and the telomerase activity can be detected in 60,000 Hela cells. This method is simple in operation and low in cost, but the detection sensitivity needs to be further improved.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O631;O629.8
本文编号：2179864
[Abstract]:Telomerase is a ribonucleoprotease composed of RNA and protein with reverse transcriptional activity. When telomerase binds to telomere DNA, it uses its own RNA sequence as a template for continuous replication to prevent telomere DNA from shortening the end during cell division. Telomerase activity is difficult to detect in human normal cells and is generally activated in tumors or cancer cells. The strong activity of tumor or cancer cells is closely related to the activation of telomerase. The detection of telomerase activity can realize the early diagnosis of tumor or cancer, and it is of great significance for the development of anticancer drugs targeting telomerase. In this dissertation, a series of telomerase activity detection methods were established by using graphene oxide and cationic conjugated polymers, combined with two signal amplification techniques: constant temperature index amplification and hybrid chain reaction. The main contents are as follows: method 1: telomerase activity was detected with high sensitivity based on magnetic graphene oxide and isothermal index amplification reaction. Telomerase extension products are DNAs with-(ggttag) n repeats. A specific DNA probe, (ctaacc) 2, which matched two repeat sequences, was designed to hybridize with telomerase extension products to form double-stranded DNAs. Excessive DNA probe was adsorbed by magnetic graphene and removed by magnetic separation. The DNA probe captured by telomerase extension product was rapidly amplified by isothermal index amplification reaction (IEXPAR). The amplified products were detected by real-time fluorescence with SYBR Green I dyes which were specifically bound to double-stranded DNA. Telomerase activity can be detected in as low as 50 cancer cells under optimal conditions. The high sensitivity detection of telomerase activity under constant temperature amplification was realized. Method two: telomerase activity was detected based on fluorescence resonance energy transfer of cationic conjugated polymer. Based on the fluorescence resonance energy transfer between cationic conjugated polymer (PFP) and fluorescent dye SYBR Green I (SG), a simple and rapid method for detecting telomerase activity was developed. In the presence of telomerase, the primer probe was extended to produce DNAs with-(ggttag) n repeats. The telomerase extension product was then connected to the magnetic microspheres by the specific interaction between streptavidin and biotin. A probe matching telomerase extension product, (ctaacc) 2, was added. Telomerase extension products can be specifically embedded into the double stranded structure of DNA after the addition of SG.SG after the formation of double strand structure. Finally, the addition of PFP.PFP binds to double-stranded DNA by electrostatic interaction, and generates fluorescence resonance energy transfer (FRET). With SG embedded in double-stranded structure. Therefore, the quantitative detection of telomerase activity can be realized according to the efficiency of FRET. The method can detect telomerase activity extracted from 300, 000 Hela cells. In order to further improve the sensitivity of the method, we combine the method with hybrid chain reaction (HCR) to amplify the detection signal. The sensitivity is increased by an order of magnitude, and the telomerase activity can be detected in 60,000 Hela cells. This method is simple in operation and low in cost, but the detection sensitivity needs to be further improved.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O631;O629.8
【参考文献】
相关硕士学位论文 前1条
1 焦肖霞;利用滚环扩增反应检测端粒酶活性及单核苷酸多态性[D];河北大学;2010年
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