利用植物表达药用干扰小RNA的研究

发布时间：2018-09-01 19:41

【摘要】：研究背景：目前在生物制药中用到的生物反应器包括细菌表达系统、酵母表达系统、昆虫细胞表达系统、哺乳动物细胞表达系统和植物表达系统,其中植物生物反应器以其技术操作简单、安全环保、经济成本低、不携带病原等优势越来越受到人们的关注。部分植物源药用蛋白已开始进入市场。但是植物中外源蛋白表达量低和不能表达高度人源化糖蛋白,限制了它的应用和发展。目前的生物药品主要是多肽和蛋白类药物,其中一些结构复杂的药物蛋白,一方面难以准确定位到靶点位置,另一方面存在空间位阻,不能与靶点高效结合发挥功能。因此,近年来核酸类小分子药物开始崭露头角,尤其是RNA干扰治疗中使用的干扰小RNA。RNA干扰(RNA interference, RNAi)中常用的干扰小RNA可分为siRNA(short interfering RNA) 和 miRNA (microRNA),前者为外源性,后者为内源性。但是二者功能一致,通过序列互补,特异性地识别靶基因mRNA使其降解或翻译抑制,从而“沉默”基因表达,这在细胞的基本活动中扮演着重要角色。RNAi在基因治疗研究中有着独特的优势：与传统药物相比,RNAi药物在疾病治疗中具有高效性、特异性、低毒性和可传递性等优点。目前RNAi治疗中使用的干扰小RNA主要由化学方法合成,由于其不稳定、易降解等特性,需要一定的化学修饰,导致化学合成的siRNA成本较高。另外一个限制RNAi药物临床应用的主要因素是RNAi药物在体内的输送,即如何将药物靶向输送到特定的病灶组织或器官。2011年,我校研究团队在Cell Research发表的研究结果显示：在哺乳动物血清中可检测出多种植物内源miRNA,其中miR168a可特异性靶向小鼠基因下调其肝脏中LDLRAP1的表达,从而说明食物来源的植物miRNA可能有跨界调控哺乳动物基因表达的作用。研究目的与意义：基于以上研究成果,我们提出利用植物表达有治疗功能的干扰小RNA,以望通过食物摄入的方式进入人或动物体内,发挥基因沉默的功能,达到基因治疗的效果。这项研究不仅解决了目前RNAi在基因治疗中遇到的问题,而且拓展了植物生物反应器在生物制药中的应用空间。研究方法与结果：本课题以HCV (hepatitis C Virus)基因组以及与2型糖尿病密切相关的PGC1αa mRNA为靶点,依据序列互补原则设计了药用干扰小RNA或称人造小RNA (artificial microRNA)的序列。然后构建表达载体质粒,通过农杆菌介导的转化技术将其转入生菜,用RT-PCR和Northern blot的方法对转化再生植株中干扰小RNA的表达进行分子鉴定。结果成功获得了阳性转化再生植株。但是还需后续动物实验或临床试验来验证生菜表达干扰小RNA的基因沉默效果和RNA干扰治疗效果。
[Abstract]:Background: bioreactors used in biopharmaceuticals include bacterial expression system, yeast expression system, insect cell expression system, mammalian cell expression system and plant expression system. Among them, plant bioreactor has attracted more and more attention because of its advantages of simple operation, safe and environmental protection, low economic cost and no pathogen. Some plant-derived medicinal proteins have begun to enter the market. However, the low expression of exogenous proteins in plants and their inability to express highly humanized glycoproteins limit its application and development. At present, biopharmaceuticals are mainly polypeptide and protein drugs, some of which have complex structure. On the one hand, it is difficult to locate the target position accurately, on the other hand, there is a space resistance, so it can not function efficiently with the target. Therefore, in recent years, nucleic acid small molecular drugs have begun to emerge, especially in the treatment of RNA interference small RNA.RNA interference (RNA interference, RNAi) commonly used interference small RNA can be divided into siRNA (short interfering RNA) and miRNA (microRNA), the former is exogenous, the latter is endogenous. But they have the same function. They can specifically recognize the target gene mRNA to degrade or inhibit their translation through sequence complementation, thus "silencing" gene expression. RNAi plays an important role in the basic activities of cells. RNAi has unique advantages in gene therapy: compared with traditional drugs, RNAi has the advantages of high efficiency, specificity, low toxicity and transmissibility in the treatment of diseases. At present, the small interfering RNA used in the treatment of RNAi is mainly synthesized by chemical method. Because of its unstable and degradable properties, it needs some chemical modification, which leads to the high cost of siRNA synthesis. Another major limiting factor in the clinical use of RNAi drugs is the delivery of RNAi drugs in vivo, that is, how to target the delivery of drugs to specific lesion tissues or organs. The results of the study published by our team in Cell Research showed that multiple plant endogenous miRNA, could be detected in mammalian serum in which miR168a could specifically target mouse genes to down-regulate the expression of LDLRAP1 in the liver. It is suggested that miRNA from food source may have the function of regulating mammalian gene expression in a cross-border manner. Objective and significance: based on the above research results, we propose to use plant expressed interference small RNA, to enter human or animal body through food intake, to play the role of gene silencing, and to achieve the effect of gene therapy. This study not only solves the problems encountered by RNAi in gene therapy, but also expands the application space of plant bioreactor in biopharmaceuticals. Methods and results: using HCV (hepatitis C Virus) genome and PGC1 伪 a mRNA closely related to type 2 diabetes mellitus as targets, the sequence of small interfering RNA or artificial small RNA (artificial microRNA) was designed according to the principle of sequence complementarity. Then the expression vector plasmid was constructed and transformed into lettuce by Agrobacterium tumefaciens-mediated transformation. The expression of interfering small RNA in regenerated plants was identified by RT-PCR and Northern blot. Results the positive transformed plants were successfully obtained. But further animal experiments or clinical trials are needed to verify the gene silencing effect of lettuce expression interfering small RNA and the therapeutic effect of RNA interference.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R915;Q789

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