功能DNA纳米材料的组装及肿瘤细胞检测研究

发布时间：2018-05-14 18:35

本文选题：DNA纳米结构 + 癌细胞　；参考：《山东大学》2017年硕士论文

【摘要】：癌症是危害全人类健康和生命安全的重大疾病。目前,研究表明早期诊断和治疗能够提高恶性肿瘤的临床疗效、降低其死亡率。因此,肿瘤发生早期的灵敏和准确检测具有十分重要的现实意义。传统检测癌症的方法在特异性、灵敏度等方面仍存在不足,无法满足肿瘤早期检测的要求。近年来,一系列针对肿瘤标志物(如mRNA、膜蛋白等)的新型检测分子不断被开发出来,为肿瘤早期诊断提供了新的机遇。DNA纳米技术自提出以来,因具有结构可编程性、良好的生物相容性和生物稳定性、无明显的细胞毒性和免疫刺激性、能够自主入胞等特点,在生物医药领域引起了广泛的关注,已经用于肿瘤的成像与检测。基于以上论述,本文利用DNA纳米技术构建了新型肿瘤检测系统,用于癌细胞中mRNA的逻辑检测以及膜蛋白免标记、信号放大型检测,实现了肿瘤细胞的灵敏、准确检测,以期为临床恶性肿瘤的早期检测提供依据。本论文构建了双识别探针共轭的DNA四面体通过mRNA的原位逻辑检测实现HepG2细胞的准确检测。该结构包括:(1)逻辑识别两种mRNA的探针。只有两种特定的mRNA——GalNAc-T mRNA和TK1 mRNA同时存在的基础上才能输出一种荧光信号,一种或者没有指定mRNA存在的情况下就没有荧光信号,准确区分肝癌细胞HepG2(存在GalNAc-TmRNA和TK1 mRNA两种mRNA)与正常肝细胞HL7702(只存在GalNAc-T mRNA);(2)DNA四面体结构。作为探针的载体,生物相容性好、易合成与修饰、结构稳定以及能够自主入胞等,具有运载探针入胞以及保护探针的作用。最终成功实现两种细胞的区分,避免了只检测一种肿瘤相关mRNA存在的假阳性现象。同时采用逻辑输出方式,结果简单直观,避免多色检测方式会受到荧光光谱重叠以及检测通道数目等的限制。本论文还构建了共区域化适体探针膜上触发信号放大装置用于Hela细胞的免标记、灵敏检测。在所设计的共区域化适体探针的基础上,结合经典的HCR反应膜上触发信号放大装置——DNA纳米条带,在生成的HCR双链部分插入Eve Green染料就可以输出荧光信号了,多余未反应完的探针可以被加入的石墨烯吸附掉,其生成的背景信号。检测的有效细胞个数为100个。另外这种策略是在识别与信号放大过程后插入染料,具有不干扰识别与信号放大过程的优点。
[Abstract]:Cancer is a major disease that endangers the health and safety of all mankind. At present, studies have shown that early diagnosis and treatment can improve the clinical efficacy of malignant tumors and reduce their mortality. Therefore, the sensitive and accurate detection of early tumorigenesis is of great practical significance. The traditional methods of cancer detection are still insufficient in specificity and sensitivity, and can not meet the requirements of early detection of cancer. In recent years, a series of new detection molecules for tumor markers (such as mRNAs, membrane proteins, etc.) have been developed, which provide a new opportunity for early diagnosis of tumor. Because of its good biocompatibility and stability, no obvious cytotoxicity and immune irritation, and the ability to enter the cell independently, it has attracted wide attention in the field of biomedicine and has been used in tumor imaging and detection. Based on the above discussion, a new tumor detection system was constructed by using DNA nanotechnology, which can be used for the logical detection of mRNA in cancer cells, as well as for the detection of membrane proteins without labeling and signal amplification, so as to realize the sensitive and accurate detection of tumor cells. In order to provide basis for early detection of clinical malignant tumor. In this paper, a conjugated DNA tetrahedron with double recognition probe was constructed to detect HepG2 cells accurately by in situ logical detection of mRNA. The structure consists of 1: 1) logical recognition of two mRNA probes. Only when two specific mRNA--GalNAc-T mRNA and TK1 mRNA exist at the same time can one kind of fluorescence signal be outputted, and there is no fluorescence signal in the presence of one or no specified mRNA. To distinguish HepG2 (GalNAc-TmRNA and TK1 mRNA) from HL7702 (only GalNAc-T mRNA2) tetrahedron in hepatoma cell line (HepG2) and normal hepatocyte (HL7702). As the carrier of the probe, it has good biocompatibility, easy synthesis and modification, stable structure and ability to enter the cell independently. It has the function of carrying the probe into the cell and protecting the probe. Finally, the differentiation of two kinds of cells was successfully realized, avoiding the false positive phenomenon of detecting only one tumor associated mRNA. At the same time, the method of logical output is used, the result is simple and intuitionistic, and the multi-color detection method is avoided by the limitation of fluorescence spectrum overlap and the number of detection channels. In this paper, we also constructed a co-regionalized aptamer probe on the membrane trigger signal amplification device for Hela cell labeling, sensitive detection. On the basis of the designed co-regionalized aptamer probe and the classic trigger signal amplification device on the HCR reaction film, the fluorescent signal can be output by inserting the Eve Green dye into the generated HCR double strand. The superfluous unreacted probe can be adsorbed by the added graphene and the generated background signal. The number of effective cells detected was 100. In addition, this strategy is to insert dyes after the recognition and signal amplification process, which has the advantage of not interfering with the process of recognition and signal amplification.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.4

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