【摘要】：在哺乳動(dòng)物的體內(nèi)有三種不同的DNA甲基轉(zhuǎn)移酶,它們分別是DNMT1,DNMT3A和DNMT3B。其中,含量最豐富的是DNMT1,被認(rèn)為是哺乳動(dòng)物中最關(guān)鍵的甲基轉(zhuǎn)移酶。實(shí)驗(yàn)表明DNA甲基轉(zhuǎn)移酶活性的異常與許多類(lèi)型的遺傳缺陷和眾多的惡性腫瘤密切相關(guān),DNA甲基轉(zhuǎn)移酶活性的異常會(huì)導(dǎo)致異常的DNA甲基化。目前,許多研究結(jié)果已經(jīng)證明,低甲基化和超甲基化存在于多種癌癥中,像宮頸癌、卵巢癌、肺癌、結(jié)腸癌、泌尿道癌、白血病等。胞嘧啶的DNA的甲基化是最常見(jiàn)的甲基化形式,其過(guò)程是在DNA甲基轉(zhuǎn)移酶催化下,將S-腺苷甲硫氨酸中的甲基基團(tuán)轉(zhuǎn)移到CpG位點(diǎn)的胞嘧啶上,該甲基化發(fā)生在嘧啶環(huán)的C5位上。最近研究表明了甲基轉(zhuǎn)移酶活性的異常通常比惡性腫瘤其他癥狀的發(fā)生要早許多,因此DNA甲基轉(zhuǎn)移酶是一種潛在的可預(yù)測(cè)的生物標(biāo)記物,可作為不同類(lèi)型的癌癥的診斷和治療過(guò)程中的藥物作用靶點(diǎn)。因此,發(fā)展簡(jiǎn)單、快速、靈敏且可用于實(shí)際樣品中甲基轉(zhuǎn)移酶活性的方法有著重要的意義。電致化學(xué)發(fā)光檢測(cè)法將電化學(xué)檢測(cè)和化學(xué)發(fā)光檢測(cè)結(jié)合在一起,和化學(xué)發(fā)光法相比,它無(wú)需發(fā)射光源,實(shí)驗(yàn)背景信號(hào)較低。具有高靈敏度、易操作、儀器易于小型化等優(yōu)點(diǎn)。本文利用電致化學(xué)發(fā)光法,實(shí)現(xiàn)了癌癥細(xì)胞中的人類(lèi)DNA甲基轉(zhuǎn)移酶活性高靈敏的檢測(cè)。本課題旨在建立對(duì)某些疾病早期細(xì)胞的甲基化水平檢測(cè)的方法,為早期的診斷和治療提供理論依據(jù)。1.我們建立了電致化學(xué)發(fā)光(ECL)生物傳感器來(lái)檢測(cè)癌細(xì)胞中甲基轉(zhuǎn)移酶(DNMT1)活性。在這個(gè)工作中,MPA修飾的摻Eu3+的CdS量子點(diǎn)(MPA-CdS:Eu NCs)作為電致化學(xué)發(fā)光的發(fā)射體。修飾了 MPA-CdS:EuNCs的玻碳(GCE)電極上連接半甲基化的雙鏈DNA,當(dāng)與DNMT1和SAM作用后,半甲基化序列5'-CGCGCG-3'的胞嘧啶C被甲基化,形成全甲基化雙鏈DNA,之后再與限制性核酸內(nèi)切酶BssHⅡ作用。經(jīng)過(guò)BssHⅡ剪切后,半甲基化的雙鏈DNA上剩余的3'端凹陷的部分能夠繼續(xù)被ExoⅢ消化掉。留在電極上的單鏈DNA部分會(huì)與prime DNA進(jìn)行雜交,這樣就引發(fā)了一個(gè)雜交鏈反應(yīng)。形成一個(gè)G-四聯(lián)體/血紅素DNA酶的超級(jí)雙鏈DNA結(jié)構(gòu),大大降低ECL的信號(hào)。因?yàn)锽ssHⅡ只能對(duì)半甲基化的序列進(jìn)行剪切,因此,甲基轉(zhuǎn)移酶的活性越高,全甲基化雙鏈DNA越多,被剪切的越少�；谏鲜龅倪^(guò)程,實(shí)現(xiàn)了對(duì)檢測(cè)細(xì)胞中的DNMT1活性的高靈敏度檢測(cè)。實(shí)驗(yàn)結(jié)果表明,電致化學(xué)發(fā)光強(qiáng)度差(全甲基化之前與形成G-四聯(lián)體的DNA酶之后的強(qiáng)度差)與DNMT1的活性有很好的線(xiàn)性,在信噪比為3時(shí),檢測(cè)限可達(dá)到0.09 U/mL,而且該生物傳感器可應(yīng)用于人肺癌細(xì)胞(A549)中DNMT1的檢測(cè),它的檢測(cè)限可達(dá)到2個(gè)A549細(xì)胞。為了驗(yàn)證該實(shí)驗(yàn)方法在臨床上的應(yīng)用,進(jìn)一步檢測(cè)了另外兩種癌癥細(xì)胞(人乳腺癌細(xì)胞MCF-7和人宮頸癌細(xì)胞Hela)和一種正常的人胚腎細(xì)胞(HKE-293)中的DNMT1的活性。結(jié)合DNMT1標(biāo)準(zhǔn)試劑盒實(shí)驗(yàn)結(jié)果,以HKE-293為基準(zhǔn),其余細(xì)胞對(duì)DNMT1的相關(guān)性進(jìn)行換算,結(jié)果表明人類(lèi)癌癥細(xì)胞對(duì)于DNMT1是高表達(dá)的,在生物醫(yī)學(xué)的研究有著潛在價(jià)值。2.通過(guò)構(gòu)建雙信號(hào)比率計(jì)算電致化學(xué)發(fā)光生物傳感器來(lái)檢測(cè)人類(lèi)甲基轉(zhuǎn)移酶活性。該方法以CdS:Eu NCs和魯米諾為信號(hào)發(fā)射體,其中CdS:Eu NCs修飾在玻碳電極表面,而魯米諾存在于實(shí)驗(yàn)檢測(cè)液中。首先,半甲基化的發(fā)卡S1 DNA連接在修飾了 CdS:Eu NCs的玻碳電極上。之后將S2 DNA與Au NPs共同作用形成了 S2 DNA-AuNP的結(jié)構(gòu)與連接在GCE電極上的S1 DNA進(jìn)行雜交,形成了 S1S2/GCE的電極組裝結(jié)構(gòu)。接下來(lái),DNMT1和SAM進(jìn)行甲基化作用,其中一部分的半甲基化的雙鏈DNA全甲基化。因?yàn)橄拗菩院怂醿?nèi)切酶BssHⅡ?qū)谆舾?只能剪切半甲基化或者未甲基化的5'-GCGCGC-3'序列,此時(shí)全甲基化的雙鏈DNA不能夠被接下來(lái)的限制性?xún)?nèi)切酶BssHⅡ進(jìn)行剪切,其他沒(méi)有全甲基化的雙鏈DNA能夠被剪切。之后,與核酸外酶ExoⅢ進(jìn)行作用,BssHⅡ作用后的半甲基化DNA雙鏈結(jié)構(gòu)中鏈接了 Au NP的S1 DNA部分被消化成片段,Au NPs脫離電極�；贑dS:Eu NCs和Au NPs之間的RET-SPR作用,以及Au NPs作為生物傳感器的電傳感原件對(duì)電致化學(xué)發(fā)光劑魯米諾有優(yōu)越的催化作用,此時(shí)的CdS:Eu NCs的強(qiáng)度有部分的恢復(fù),而魯米諾的強(qiáng)度降低,通過(guò)CdS:Eu NCs和魯米諾強(qiáng)度的比值來(lái)檢測(cè)DNMT1的活性實(shí)驗(yàn)。實(shí)驗(yàn)結(jié)果表明,該方法可以用來(lái)檢測(cè)DNMT1的活性,它的最低檢測(cè)限為0.07 U/mL。比之傳統(tǒng)的單信號(hào)源方法更加的準(zhǔn)確。實(shí)驗(yàn)同樣可用于對(duì)甲基轉(zhuǎn)移酶抑制劑的評(píng)估,對(duì)抗癌藥物的發(fā)現(xiàn)有潛在的可能性。
[Abstract]:There are three different DNA methyltransferases in the mammalian body, which are DNMT1, DNMT3A and DNMT3B., which are the most abundant in DNMT1. They are considered the most critical methyltransferase in mammals. The experiment shows that the abnormal activity of DNA methyltransferase is closely related to many types of genetic defects and many malignant tumors. Abnormality in the activity of DNA methyltransferase can lead to abnormal DNA methylation. At present, many studies have shown that hyper methylation and hypermethylation exists in various cancers, such as cervical cancer, ovarian cancer, lung cancer, colon cancer, urinary tract cancer, leukemia and so on. Methylation of DNA of cytosine is the most common methylation in DNA a The methylation of S- adenosine methionine is transferred to cytosine at the CpG site, which occurs at the C5 site of the pyrimidine ring. Recent studies have shown that the abnormality of the activity of methyltransferase is usually much earlier than that of other malignant tumors, because the DNA methyltransferase is a potential predictability Biomarkers can be used as drug targets in the diagnosis and treatment of different types of cancer. Therefore, it is of great significance to develop simple, rapid, sensitive and useful methods for the activity of methyltransferase in actual samples. Electrochemiluminescence detection combines electrochemical detection with chemiluminescence detection, and chemistry. Compared with the luminescence method, it does not need to launch the light source, the experimental background signal is low. It has the advantages of high sensitivity, easy operation and easy to miniaturization. In this paper, the high sensitivity detection of human DNA methyltransferase activity in cancer cells is realized by electrochemiluminescence. This lesson is designed to establish the level of methylation of early cells in some diseases. The method of detection provides a theoretical basis for early diagnosis and treatment..1. we have established an electrochemiluminescence (ECL) biosensor to detect the activity of methyltransferase (DNMT1) in cancer cells. In this work, MPA modified Eu3+ CdS quantum dots (MPA-CdS:Eu NCs) are used as emitters for electrochemiluminescence. On the glassy carbon (GCE) electrode, the semi methylated double stranded DNA is connected to the cytosine C of the methylation sequence 5'-CGCGCG-3'after the action of DNMT1 and SAM, forming a fully methylated double stranded DNA and then acting with the restrictive endonuclease BssH II. After the BssH II shear, the remaining part of the 3' endpoint depression on the semi methylation of the double stranded DNA is possible. Continue to be digested by Exo III. The single strand DNA part on the electrode will cross with Prime DNA, thus triggering a hybrid chain reaction. Forming a super double stranded DNA structure of G- four coupling / heme DNA enzyme, greatly reducing the signal of ECL, because BssH II can only cut the sequence of hemimethylation, thus, the activity of methyltransferase The higher the sex, the more the total methylation double strand DNA, the less the shear is. Based on the above process, a high sensitivity test for the detection of DNMT1 activity in the cells is realized. The results show that the poor intensity of electrochemiluminescence (the intensity difference after the methylation with the DNA enzyme formed by the G- four coupling) is well linear with the activity of DNMT1, in the letter When the noise ratio is 3, the detection limit can reach 0.09 U/mL, and the biosensor can be applied to the detection of DNMT1 in human lung cancer cells (A549). The detection limit can reach 2 A549 cells. In order to verify the clinical application of the experimental method, the other two cancer cells (human breast cancer cell MCF-7 and human cervical cancer cell Hela) are further detected. And the activity of DNMT1 in a normal human embryonic kidney cell (HKE-293). Combined with the results of the DNMT1 standard kit experiment, on the basis of HKE-293, the rest of the cells were converted to the correlation of DNMT1. The results showed that the human cancer cells were highly expressed to DNMT1, and the potential value of.2. in biomedical research was achieved by constructing a dual signal ratio meter. The electrochemiluminescence biosensor is used to detect the activity of human methyltransferase. This method uses CdS:Eu NCs and Lumino as the signal emitter, in which CdS:Eu NCs is modified on the surface of the glassy carbon electrode and Lumino exists in the experimental detection solution. First, the semi methylation S1 DNA connected to the glassy carbon electrode of the CdS:Eu NCs. The co action of S2 DNA and Au NPs formed the structure of S2 DNA-AuNP with the S1 DNA connected to the GCE electrode and formed the electrode assembly structure of S1S2/GCE. Next, DNMT1 and SAM were methylation, in which a part of the semi methylated total methylation of S1S2/GCE was sensitive to the sensitivity of the restriction endonuclease II to methylation. It can only cut the 5'-GCGCGC-3'sequence of methylation or methylation, at this time the total methylation of the double stranded DNA can not be cut by the next restrictive endonuclease BssH II, and the other double stranded DNA without all methylation can be cut. Then, it is used with the nucleic acid outer enzyme Exo III, and the semi methylation of the DNA double stranded structure after the action of BssH II. The S1 DNA part of the Au NP is digested into fragments, Au NPs detached from the electrode. Based on the RET-SPR action between CdS:Eu NCs and Au NPs, and an electrical sensing original for the biosensor as a biosensor, the CdS:Eu NCs has a superior catalytic effect on the electrochemiluminescence agent Lumino, and the intensity of Lumino is reduced in part. Test the activity of DNMT1 by the ratio of CdS:Eu NCs to Lumino strength. The experimental results show that the method can be used to detect the activity of DNMT1, and its minimum detection limit is 0.07 U/mL. more accurate than that of the traditional single signal source method. The potential potential.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R446;O657.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉姝娜;屠蘊(yùn)秋;李文;吳萍;張卉;蔡稱(chēng)心;;脫氧核糖核酸甲基化分析方法在腫瘤診斷和治療中的應(yīng)用[J];分析化學(xué);2011年09期

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本文編號(hào)：2159373