【摘要】：端粒酶是由RNA和蛋白質(zhì)組成的核糖核蛋白酶,具有反轉(zhuǎn)錄活性。端粒酶與端粒DNA結(jié)合后,會(huì)以自身RNA序列為模板,進(jìn)行不斷的復(fù)制,阻止端粒DNA在細(xì)胞分裂過(guò)程中造成的末端縮短。端粒酶在人的正常細(xì)胞中很難被檢測(cè)到活性,它一般在腫瘤或癌細(xì)胞中被激活。腫瘤或癌細(xì)胞的強(qiáng)大活性與端粒酶被激活密切相關(guān)。通過(guò)檢測(cè)端粒酶活性可以實(shí)現(xiàn)腫瘤或癌癥的早期診斷,同時(shí)對(duì)開發(fā)以端粒酶為靶標(biāo)分子的抗癌藥物具有重要意義。本學(xué)位論文分別利用氧化石墨烯和陽(yáng)離子共軛聚合物,結(jié)合恒溫指數(shù)擴(kuò)增和雜交鏈?zhǔn)椒磻?yīng)兩種信號(hào)放大技術(shù),建立了系列端粒酶活性檢測(cè)的方法。主要研究?jī)?nèi)容如下:方法一:基于磁性氧化石墨烯和恒溫指數(shù)擴(kuò)增反應(yīng)高靈敏度檢測(cè)端粒酶活性。端粒酶延伸產(chǎn)物是具有-(ggttag)n重復(fù)序列的DNA。設(shè)計(jì)一條與兩個(gè)重復(fù)序列相匹配的特異性的DNA探針-(ctaacc)2使其與端粒酶延伸產(chǎn)物雜交,形成雙鏈DNA。過(guò)量的DNA探針被磁性石墨烯吸附,通過(guò)磁分離去除。利用恒溫指數(shù)擴(kuò)增反應(yīng)(IEXPAR)對(duì)端粒酶延伸產(chǎn)物捕捉的DNA探針進(jìn)行快速的擴(kuò)增。用與雙鏈DNA特異性結(jié)合的SYBR Green I染料對(duì)擴(kuò)增產(chǎn)物進(jìn)行實(shí)時(shí)熒光檢測(cè)。在最優(yōu)條件下,可以檢測(cè)到低至50個(gè)癌細(xì)胞中的端粒酶活性。實(shí)現(xiàn)了恒溫?cái)U(kuò)增條件下端粒酶活性的高靈敏度檢測(cè)。方法二:基于陽(yáng)離子共軛聚合物熒光共振能量轉(zhuǎn)移檢測(cè)端粒酶活性。基于陽(yáng)離子共軛聚合物(PFP)與熒光染料SYBR Green I(SG)之間的熒光共振能量轉(zhuǎn)移,建立了一種簡(jiǎn)單快速的端粒酶活性檢測(cè)方法。當(dāng)端粒酶存在時(shí),引物探針被延伸,生成具有-(ggttag)n重復(fù)序列的DNA。然后通過(guò)鏈霉親合素與生物素的特異性作用將端粒酶延伸產(chǎn)物連接在磁性微球上。加入與端粒酶延伸產(chǎn)物匹配的探針-(ctaacc)2。端粒酶延伸產(chǎn)物形成雙鏈結(jié)構(gòu)之后加入SG。SG能夠特異性的嵌入到DNA雙鏈結(jié)構(gòu)中。最后,加入PFP。PFP通過(guò)靜電作用與雙鏈DNA結(jié)合,與嵌入在雙鏈結(jié)構(gòu)中的SG發(fā)生熒光共振能量轉(zhuǎn)移(FRET)。因此,根據(jù)FRET的效率可以實(shí)現(xiàn)對(duì)端粒酶活性的定量檢測(cè)。該方法可以檢測(cè)到30萬(wàn)個(gè)Hela細(xì)胞中提取的端粒酶活性。為了進(jìn)一步提高方法的靈敏度,我們將該方法與雜交鏈?zhǔn)椒磻?yīng)(HCR)結(jié)合,實(shí)現(xiàn)了檢測(cè)信號(hào)的放大,靈敏度提高了一個(gè)數(shù)量級(jí),可以檢測(cè)到6萬(wàn)個(gè)Hela細(xì)胞中的端粒酶活性。此方法操作簡(jiǎn)單,檢測(cè)成本低,但檢測(cè)靈敏度還有待于進(jìn)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.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O631;O629.8

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相關(guān)碩士學(xué)位論文 前1條

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本文編號(hào)：2179864