基于配体诱导的人工核酶开关对哺乳动物细胞基因表达调控的研究

发布时间：2018-03-06 17:09

本文选题：配体诱导　切入点：人工核酶开关　出处：《中国科学技术大学》2017年博士论文　论文类型：学位论文

【摘要】：近些年合成生物学的研究建立了一系列的能够在细胞内调节基因表达的非编码RNA控制的体系,其中核糖开关,是一类广泛存在于各种生物体内并对小分子代谢物敏感的结构RNA。它们可以不依赖任何蛋白因子,直接结合小分子代谢物,形成选择性茎环结构或通过自我剪切功能,从转录或翻译水平来调控基因表达。据此,将体外筛选出的适体区序列和一些天然核酶的序列相融合构建人工核酶开关,可以利用配体和适体的结合导致的构象改变核酶自我剪切的活性,进而调控相关基因的表达,从而可被运用于包括哺乳动物细胞在内的各种生物机体中。近些年,核酶开关也被用于疾病治疗的研究中,但是将这种基于人工RNA的调节系统对于哺乳细胞内功能基因的作用在临床研究还面临诸多挑战。在肿瘤细胞基因治疗中,HSV-TK/GCV系统与RNA干扰是最为常用的治疗方法。本研究首先以肿瘤细胞为模型,构建了基于茶碱小分子激活的的锤头状核酶开关,建立了稳定转染该系统的HeLa细胞系,利用核酶开关与低毒性小分子配体的结合,调控核酶的剪切,实现对HSV-TK基因mRNA的顺式调节,建立了可逆的、长期的、稳定的低毒性基因调控系统。随后,我们构建了基于配体作用的HDV核酶开关调节系统,通过响应茶碱分子浓度变化调节剪切,精确释放靶基因的pri-miRNA,实现对肿瘤细胞抗凋亡基因Bcl-2的RNA干扰作用的实时有效调控,为今后RNA干扰在肿瘤细胞中的基因治疗更安全有效的运用提供理论基础与研究方法。此外,本研究通过核酶开关的构建,在真核动物细胞中建立了硫胺素焦磷酸(TPP)浓度响应的EGFP荧光传感器,可将其浓度的变化转化为报告基因表达的改变,发展出对哺乳活细胞内代谢物或因子的无标记、无损伤、可视、高效的检测方法。
[Abstract]:In recent years, synthetic biology has established a series of non-coding RNA control systems that can regulate gene expression in cells. RNAs are a class of structures that are widely present in a variety of organisms and are sensitive to small molecular metabolites. They can bind directly to small molecular metabolites without any protein factor, forming selective stem-ring structures or through self-cutting functions. Gene expression is regulated by transcription or translation. Based on this, artificial ribozyme switch is constructed by fusion of aptamer region sequence selected in vitro with some natural ribozyme sequence. The conformation resulting from the binding of ligands and aptamers can be used to alter the ribozyme self-cleavage activity, thereby regulating the expression of related genes, which can be used in a variety of organisms, including mammalian cells. Ribozyme switches have also been used in disease treatment studies, However, there are still many challenges in clinical research on the role of this regulation system based on artificial RNA in mammalian intracellular functional genes. HSV-TK / GCV system and RNA interference are the most commonly used methods in tumor cell gene therapy. In this study, tumor cells were first used as models. A hammerhead ribozyme switch based on theophylline small molecule activation was constructed and a HeLa cell line stably transfected with theophylline was established. The ribozyme shearing was regulated by the combination of ribozyme switch and low toxic small molecule ligand. The cis regulation of HSV-TK gene mRNA was realized, and a reversible, long-term and stable low toxicity gene regulation system was established. Subsequently, we constructed a HDV ribozyme switching regulatory system based on ligand interaction. By responding to the variation of theophylline molecular concentration to regulate the shearing and release the pri-miRNAs of the target gene accurately, the real-time and effective regulation of RNA interference on the anti-apoptotic gene Bcl-2 in tumor cells was realized. This study provides a theoretical basis and research method for the safe and effective use of RNA interference in gene therapy in tumor cells in the future. In addition, the construction of ribozyme switch is used in this study. A EGFP fluorescence sensor for the concentration response of thiamine pyrophosphate (TPP) was established in eukaryotic animal cells. The change of thiamine pyrophosphate concentration could be transformed into the change of reporter gene expression, and no labeling or damage to metabolites or factors in mammalian living cells was developed. Visual, efficient detection method.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q78

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