临床肝癌组织核酸适配体的筛选及初步应用研究

发布时间：2018-03-30 11:50

本文选题：核酸适配体　切入点：筛选　出处：《湖南大学》2016年硕士论文

【摘要】：肝癌是当今全世界发病率及致死率最高的癌症之一。发展新型高效、快速的肿瘤标志物筛选方法,以及开发对肿瘤病理组织具有高特异性、高灵敏度并且化学性质稳定的分子识别探针对于临床肿瘤诊断、分类及判断预后等具有非常重要意义。近年来,基于protein-SELEX或者cell-SELEX技术筛选得到的核酸适配体作为一种新发展起来的识别分子在包括癌症研究的许多领域获得了广泛的应用。但是protein-SELEX方法的筛选靶标为纯化蛋白,而cell-SELEX技术以体外培养的肿瘤细胞系为筛选靶标,这些筛选目标物都无法真正模拟体内肿瘤的实际发生发展模式,忽略了肿瘤形成过程中会受到体内包括血管微环境和细胞外周基质等的多种因素影响,这使得基于cell-SELEX筛选得到的核酸适配体一般只能识别体外培养的肿瘤细胞系,难以真正实现对肿瘤临床病理组织的检测和诊断,严重限制了核酸适配体在肿瘤临床领域的研究和应用。而tissue-SELEX技术以临床肿瘤组织为筛选靶标,能获得真正识别临床肿瘤病理组织的核酸适配体探针,在肿瘤标志物的发现、临床诊断和分型研究中展示出了良好的潜力,但是相关研究目前还开展得太少,有待进一步推进。本论文采用tissue-SELEX技术,以临床肝癌组织切片为样本,成功筛选得到能区分肝癌组织与正常肝组织的核酸适配体,并对核酸适配体与靶标的结合性能进行了分析研究。(1)基于tissue-SELEX技术,以临床肝癌组织切片为靶标,癌旁正常肝组织为对照进行筛选。此外,我们还对筛选方法进行了修改,利用前人筛选得到的核酸适配体BC15对组织中的相关位点进行了封闭,希望筛选得到的核酸适配体结合一个新位点。经过11轮不断增加筛选压力的反复筛选过程,成功鉴定出一条能区分肝癌组织与正常肝组织的核酸适配体SW1。(2)用固定的癌细胞系为模型,通过荧光共定位表征发现核酸适配体SW1的靶分子亚细胞定位在细胞核,而且不是核酸,可能是一种核酸结合蛋白；利用流式细胞术,我们测定出SW1对肝癌SMMC-7721细胞的解离常数在纳摩尔级别,展现出较强的亲和力；竞争实验表明我们筛选得到的核酸适配体SW1与其他研究组筛选得到的核酸适配体BC15的识别位点不相同。此外,通过组织芯片考察,核酸适配体对临床肝癌组织的识别率达到了72.7%,且对来源于多种器官的肿瘤组织都有较强的结合能力。综合以上分析结果,我们筛选得到的核酸适配体SW1能区分肝癌组织与正常肝组织,对于肝癌的临床筛查和诊断有一定的辅助指导价值,而且由于SW1的靶分子为一种亚细胞定位在细胞核的蛋白质,因此SW1有望用于鉴定新的肿瘤标志物,对于开发早期诊断和精准治疗方法,以及研究肝癌的发病机理有积极推动作用。
[Abstract]:Liver cancer is one of the highest morbidity and fatality rates in the world. The development of new and rapid screening methods for tumor markers and the development of high specificity for tumor pathological tissue. Highly sensitive and chemically stable molecular recognition probes are of great significance for clinical tumor diagnosis, classification and prognosis. Nucleic acid aptamers screened based on protein-SELEX or cell-SELEX technology have been widely used in many fields including cancer research as a newly developed recognition molecule. But the screening target of protein-SELEX method is purified protein. However, cell-SELEX technology uses tumor cell lines cultured in vitro as the screening target, and none of these screening targets can really mimic the actual occurrence and development pattern of tumor in vivo. Many factors, including vascular microenvironment and peripheral matrix, have been neglected in tumor formation, which makes the aptamers selected based on cell-SELEX can only recognize tumor cell lines in vitro. It is difficult to detect and diagnose tumor clinicopathological tissue, which seriously limits the research and application of aptamer in the clinical field of tumor. However, tissue-SELEX technology selects clinical tumor tissue as the target. The aptamer probe that can truly identify clinical tumor tissues shows good potential in the discovery of tumor markers, clinical diagnosis and typing, but the relevant studies are still too few. In this paper, tissue-SELEX technique was used to select the nucleic acid aptamers which could distinguish the liver cancer tissue from the normal liver tissue, and the clinical liver tissue section was used as the sample to screen the aptamer of nucleic acid. The binding properties of aptamers to target were analyzed and studied. (1) based on tissue-SELEX technique, clinical liver cancer tissue sections were used as target and normal liver tissues adjacent to cancer were selected as control. In addition, we modified the screening method. The aptamer BC15 was used to block the relevant sites in the tissue and to bind the aptamer to a new site. After 11 rounds of repeated screening with increasing screening pressure, A nucleic acid aptamer SW1.2capable of distinguishing liver cancer tissue from normal liver tissue was successfully identified. Using a fixed cancer cell line as a model, the target subcell of aptamer SW1 was found to be located in the nucleus and not nucleic acid by fluorescence co-localization. Using flow cytometry, we determined that the dissociation constant of SW1 to SMMC-7721 cells of liver cancer showed strong affinity at the nanomolor level. Competitive experiments showed that the nucleic acid aptamer SW1 we screened was different from the nucleic acid aptamer BC15 selected by other team. The recognition rate of aptamer of nucleic acid to clinical liver cancer tissue is 72.7, and it has strong binding ability to tumor tissue from many organs. The nucleic acid aptamer SW1 we screened can distinguish the liver cancer tissue from the normal liver tissue, which is helpful for clinical screening and diagnosis of liver cancer. Moreover, the target molecule of SW1 is a subcellular protein located in the nucleus. Therefore, SW1 is expected to be used to identify new tumor markers, which can promote the development of early diagnosis and accurate treatment, as well as the study of pathogenesis of liver cancer.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

【相似文献】