电致化学发光法检测人类甲基转移酶（DNMT1）活性的研究
发布时间：2018-08-02 12:04
【摘要】：在哺乳动物的体内有三种不同的DNA甲基转移酶,它们分别是DNMT1,DNMT3A和DNMT3B。其中,含量最丰富的是DNMT1,被认为是哺乳动物中最关键的甲基转移酶。实验表明DNA甲基转移酶活性的异常与许多类型的遗传缺陷和众多的恶性肿瘤密切相关,DNA甲基转移酶活性的异常会导致异常的DNA甲基化。目前,许多研究结果已经证明,低甲基化和超甲基化存在于多种癌症中,像宫颈癌、卵巢癌、肺癌、结肠癌、泌尿道癌、白血病等。胞嘧啶的DNA的甲基化是最常见的甲基化形式,其过程是在DNA甲基转移酶催化下,将S-腺苷甲硫氨酸中的甲基基团转移到CpG位点的胞嘧啶上,该甲基化发生在嘧啶环的C5位上。最近研究表明了甲基转移酶活性的异常通常比恶性肿瘤其他症状的发生要早许多,因此DNA甲基转移酶是一种潜在的可预测的生物标记物,可作为不同类型的癌症的诊断和治疗过程中的药物作用靶点。因此,发展简单、快速、灵敏且可用于实际样品中甲基转移酶活性的方法有着重要的意义。电致化学发光检测法将电化学检测和化学发光检测结合在一起,和化学发光法相比,它无需发射光源,实验背景信号较低。具有高灵敏度、易操作、仪器易于小型化等优点。本文利用电致化学发光法,实现了癌症细胞中的人类DNA甲基转移酶活性高灵敏的检测。本课题旨在建立对某些疾病早期细胞的甲基化水平检测的方法,为早期的诊断和治疗提供理论依据。1.我们建立了电致化学发光(ECL)生物传感器来检测癌细胞中甲基转移酶(DNMT1)活性。在这个工作中,MPA修饰的掺Eu3+的CdS量子点(MPA-CdS:Eu NCs)作为电致化学发光的发射体。修饰了 MPA-CdS:EuNCs的玻碳(GCE)电极上连接半甲基化的双链DNA,当与DNMT1和SAM作用后,半甲基化序列5'-CGCGCG-3'的胞嘧啶C被甲基化,形成全甲基化双链DNA,之后再与限制性核酸内切酶BssHⅡ作用。经过BssHⅡ剪切后,半甲基化的双链DNA上剩余的3'端凹陷的部分能够继续被ExoⅢ消化掉。留在电极上的单链DNA部分会与prime DNA进行杂交,这样就引发了一个杂交链反应。形成一个G-四联体/血红素DNA酶的超级双链DNA结构,大大降低ECL的信号。因为BssHⅡ只能对半甲基化的序列进行剪切,因此,甲基转移酶的活性越高,全甲基化双链DNA越多,被剪切的越少。基于上述的过程,实现了对检测细胞中的DNMT1活性的高灵敏度检测。实验结果表明,电致化学发光强度差(全甲基化之前与形成G-四联体的DNA酶之后的强度差)与DNMT1的活性有很好的线性,在信噪比为3时,检测限可达到0.09 U/mL,而且该生物传感器可应用于人肺癌细胞(A549)中DNMT1的检测,它的检测限可达到2个A549细胞。为了验证该实验方法在临床上的应用,进一步检测了另外两种癌症细胞(人乳腺癌细胞MCF-7和人宫颈癌细胞Hela)和一种正常的人胚肾细胞(HKE-293)中的DNMT1的活性。结合DNMT1标准试剂盒实验结果,以HKE-293为基准,其余细胞对DNMT1的相关性进行换算,结果表明人类癌症细胞对于DNMT1是高表达的,在生物医学的研究有着潜在价值。2.通过构建双信号比率计算电致化学发光生物传感器来检测人类甲基转移酶活性。该方法以CdS:Eu NCs和鲁米诺为信号发射体,其中CdS:Eu NCs修饰在玻碳电极表面,而鲁米诺存在于实验检测液中。首先,半甲基化的发卡S1 DNA连接在修饰了 CdS:Eu NCs的玻碳电极上。之后将S2 DNA与Au NPs共同作用形成了 S2 DNA-AuNP的结构与连接在GCE电极上的S1 DNA进行杂交,形成了 S1S2/GCE的电极组装结构。接下来,DNMT1和SAM进行甲基化作用,其中一部分的半甲基化的双链DNA全甲基化。因为限制性核酸内切酶BssHⅡ对甲基化敏感,只能剪切半甲基化或者未甲基化的5'-GCGCGC-3'序列,此时全甲基化的双链DNA不能够被接下来的限制性内切酶BssHⅡ进行剪切,其他没有全甲基化的双链DNA能够被剪切。之后,与核酸外酶ExoⅢ进行作用,BssHⅡ作用后的半甲基化DNA双链结构中链接了 Au NP的S1 DNA部分被消化成片段,Au NPs脱离电极。基于CdS:Eu NCs和Au NPs之间的RET-SPR作用,以及Au NPs作为生物传感器的电传感原件对电致化学发光剂鲁米诺有优越的催化作用,此时的CdS:Eu NCs的强度有部分的恢复,而鲁米诺的强度降低,通过CdS:Eu NCs和鲁米诺强度的比值来检测DNMT1的活性实验。实验结果表明,该方法可以用来检测DNMT1的活性,它的最低检测限为0.07 U/mL。比之传统的单信号源方法更加的准确。实验同样可用于对甲基转移酶抑制剂的评估,对抗癌药物的发现有潜在的可能性。
[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.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446;O657.3
本文编号：2159373
[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.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446;O657.3
【参考文献】
相关期刊论文 前1条
1 刘姝娜;屠蕴秋;李文;吴萍;张卉;蔡称心;;脱氧核糖核酸甲基化分析方法在肿瘤诊断和治疗中的应用[J];分析化学;2011年09期
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