核酸适配体介导的载有阿霉素的DNA四面体对MUC1阳性肿瘤细胞的靶向杀伤作用

发布时间：2018-06-24 13:32

本文选题：MUC1 + 核酸适配体　；参考：《北京协和医学院》2016年硕士论文

【摘要】：目的：化疗是晚期肿瘤的主要治疗手段,但传统化疗药物的细胞毒性作用往往会引起全身的系统毒性反应,降低患者的耐受程度,导致治疗效果下降。靶向治疗作为一种可以与肿瘤细胞靶向结合从而抑制肿瘤生长的治疗手段,是克服传统化疗系统毒性的重要技术路径之一。MUC1是一种跨膜糖蛋白,在多种肿瘤细胞中过量表达,包括乳腺癌,肺癌,胃癌,结肠癌,前列腺癌,胰腺癌,卵巢癌等。MUC1在肿瘤的生长,代谢,转移等方面也有一定的作用。除此之外在肿瘤细胞中表达的MUC1的结构与正常组织细胞中表达的MUC1不同,其糖基化程度较低,因此使得MUC1成为了一种具有一定肿瘤特症性的靶标分子,为其在靶向治疗的应用中提供了生物学基础。已经有研究表明,针对MUC1的单克隆抗体、疫苗、小分子配体等手段在体外和体内的肿瘤抑制实验中,可取得一定的初步效果,但目前MUC1靶向治疗的临床转化依然十分遥远,因此有必要探索针对MUC1的新型靶向治疗方法。核酸适配体(aptamer)是一类由寡聚核苷酸组成的具有高特异性和亲和性的小分子配体。与抗体相比,核酸适配体具有较低的免疫原性和较强的组织穿透性,并且制备过程简便,因此具有极大应用前景。DNA正四面体(Td)作为一种DNA纳米结构,其自组装的特点使得该物质具有结构稳定、制备过程简单、容易被功能性基团修饰的优势。已有研究表明,DNA正四面体可以作为一种药物载体,可负载抗肿瘤药物阿霉素(Doxorubicin,或Dox)。然而,目前尚未有研究报道将核酸适配体与DNA正四面体相偶联构建成为靶向药物呈递系统。本研究通过将MUC1核酸适配体(Apt)与DNA正四面体相偶联,构建成为靶向性的阿霉素呈递系统,并且在体外评估了该靶向载体是否能够与MUC1阳性肿瘤细胞特异结合,以及是否能靶向性地对MUC1阳性肿瘤细胞产生细胞毒性。方法：利用碱基互补配对的原则,通过自组装的方式,构建出DNA正四面体以及与MUC1 aptamer相偶联后的Apt-Td。通过用FAM荧光染料标记Apt-Td,利用流式细胞分析技术来检测Apt-Td是否可以特异性的与MUC1阳性肿瘤细胞结合。通过物理方法,将阿霉素载入DNA纳米结构,构建成为载有阿霉素的DNA纳米结构(Apt-Dox, Td-Dox,Apt-Td-Dox),评估其载药率,并用于后续实验。利用共聚焦成像技术,流式细胞分析技术以及细胞共培养来验证Apt-Td-Dox在体外是否可以将阿霉素靶向呈递给MUC1阳性肿瘤细胞,并且通过MTS法测定Apt-Td-Dox在MUC1阳性肿瘤细胞与MUC1阴性对照细胞中产生的细胞毒性。结果：本实验利用MUC1 aptamer和DNA正四面体通过自组装的方式成功构建了目标药物载体Apt-Td,并对其进行了粒径和Zeta电位的表征,Apt-Td的平均粒径大小为12.38nm,其Zeta电位为-10.mV；流式细胞分析结果表明Apt-Td与本实验所采用的游离MUC1 aptamer类似,可以选择性地与MUC1阳性肿瘤细胞结合而基本不与MUC1阴性细胞结合；通过对三种DNA纳米结构载药率的研究发现,Apt-Td的载药率是游离aptamer载药率的25倍;细胞摄取实验表明Apt-Td-Dox可以将阿霉素靶向呈递至MUC1阳性肿瘤细胞而限制阿霉素在MUC1阴性细胞中的药物积累；细胞毒性试验表明,Apt-Td-Dox对MUC1阳性肿瘤细胞可以产生显著的细胞毒性,并同时降低阿霉素对MUC1阴性细胞的毒性(P0.01)。结论：利用MUC1 aptamer和DNA正四面体可构建成具有靶向作用的药物载体Apt-Td。体外试验显示,该药物载体能靶向性的对MUC1阳性肿瘤细胞产生细胞毒性。
[Abstract]:Objective: chemotherapy is the main treatment for advanced tumor, but the cytotoxic effect of traditional chemotherapeutic drugs often causes systemic systemic toxicity, reducing the patient's tolerance and reducing the effect of treatment. Targeted therapy is a therapeutic method which can combine the target of tumor cells to inhibit the growth of tumor. One of the most important techniques for the toxicity of the system.MUC1 is a transmembrane glycoprotein, which is overexpressed in a variety of tumor cells, including breast cancer, lung cancer, gastric cancer, colon cancer, prostate cancer, pancreatic cancer, ovarian cancer, and other aspects of the tumor growth, metabolism, and metastasis. In addition to the expression of MU in tumor cells The structure of C1 is different from the MUC1 expressed in normal tissue cells, and its glycosylation degree is low. Therefore, it makes MUC1 a target molecule with certain tumor specificity and provides a biological basis for its application in targeted therapy. In vitro and in vivo tumor inhibition experiments, some preliminary results can be obtained, but the clinical transformation of MUC1 targeting therapy is still very far away. Therefore, it is necessary to explore new targeting therapy for MUC1. Aptamer (aptamer) is a class of small molecular ligands with high specificity and affinity formed by oligonucleotides. Compared with the antibody, the aptamers have lower immunogenicity and stronger tissue penetration, and the preparation process is simple. Therefore, the.DNA tetrahedron (Td) is a kind of DNA nanostructure. Its self assembly characteristics make the material stable, simple preparation process and easy to be modified by functional groups. Potential. Studies have shown that DNA tetrahedron can be used as a drug carrier and can load the antitumor drug adriamycin (Doxorubicin, or Dox). However, there has not been a study on the coupling of aptamers with DNA tetrahedron to a targeting drug delivery system. This study was conducted by using the MUC1 aptamer (Apt) and DNA tetrahedron. Phase coupling, construction of a targeted adriamycin presentation system, and in vitro evaluation of whether the target carrier can specifically bind to MUC1 positive tumor cells and whether it can target MUC1 positive tumor cells to cytotoxicity. Method: using the principle of base complementary pairing, the DNA positive is constructed by self-assembly. Tetrahedron and Apt-Td. coupled with MUC1 aptamer are labeled Apt-Td by FAM fluorescent dye, and flow cytometry is used to detect whether Apt-Td can specifically bind to MUC1 positive tumor cells. By physical methods, adriamycin is loaded into the DNA nanostructure and is constructed as a DNA nanostructure carrying doxorubicin (Apt-Dox, T). D-Dox, Apt-Td-Dox), evaluate the drug loading rate, and use the confocal imaging technique, flow cytometry and cell co culture to verify that the target of adriamycin can be presented to MUC1 positive tumor cells in vitro, and the Apt-Td-Dox in MUC1 positive tumor cells and MUC1 negative is determined by MTS method. Results: the target drug carrier Apt-Td was successfully constructed by self assembly of MUC1 aptamer and DNA tetrahedron, and the particle size and Zeta potential were characterized. The average size of Apt-Td was 12.38nm, and its Zeta potential was -10.mV; the result of flow cytometry showed Apt-Td. Similar to free MUC1 aptamer used in this experiment, it can selectively bind to MUC1 positive tumor cells and not combine with MUC1 negative cells. By studying the drug loading rate of three DNA nanoscale structures, the drug carrying rate of Apt-Td is 25 times that of free aptamer drug loading rate; cell uptake experiments show that Apt-Td-Dox can make adriamycin. Targeting MUC1 positive tumor cells to restrict the accumulation of adriamycin in MUC1 negative cells; cytotoxicity test showed that Apt-Td-Dox could produce significant cytotoxicity to MUC1 positive tumor cells and decreased the toxicity of adriamycin to MUC1 negative cells (P0.01). Conclusion: MUC1 aptamer and DNA positive tetrahedron can be used. The targeting drug delivery vector Apt-Td. in vitro showed that the drug carrier could be cytotoxic to MUC1 positive tumor cells.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R730.5

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