本文選題：配體誘導(dǎo)　切入點(diǎn)：人工核酶開關(guān)　出處：《中國科學(xué)技術(shù)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：近些年合成生物學(xué)的研究建立了一系列的能夠在細(xì)胞內(nèi)調(diào)節(jié)基因表達(dá)的非編碼RNA控制的體系,其中核糖開關(guān),是一類廣泛存在于各種生物體內(nèi)并對小分子代謝物敏感的結(jié)構(gòu)RNA。它們可以不依賴任何蛋白因子,直接結(jié)合小分子代謝物,形成選擇性莖環(huán)結(jié)構(gòu)或通過自我剪切功能,從轉(zhuǎn)錄或翻譯水平來調(diào)控基因表達(dá)。據(jù)此,將體外篩選出的適體區(qū)序列和一些天然核酶的序列相融合構(gòu)建人工核酶開關(guān),可以利用配體和適體的結(jié)合導(dǎo)致的構(gòu)象改變核酶自我剪切的活性,進(jìn)而調(diào)控相關(guān)基因的表達(dá),從而可被運(yùn)用于包括哺乳動物細(xì)胞在內(nèi)的各種生物機(jī)體中。近些年,核酶開關(guān)也被用于疾病治療的研究中,但是將這種基于人工RNA的調(diào)節(jié)系統(tǒng)對于哺乳細(xì)胞內(nèi)功能基因的作用在臨床研究還面臨諸多挑戰(zhàn)。在腫瘤細(xì)胞基因治療中,HSV-TK/GCV系統(tǒng)與RNA干擾是最為常用的治療方法。本研究首先以腫瘤細(xì)胞為模型,構(gòu)建了基于茶堿小分子激活的的錘頭狀核酶開關(guān),建立了穩(wěn)定轉(zhuǎn)染該系統(tǒng)的HeLa細(xì)胞系,利用核酶開關(guān)與低毒性小分子配體的結(jié)合,調(diào)控核酶的剪切,實(shí)現(xiàn)對HSV-TK基因mRNA的順式調(diào)節(jié),建立了可逆的、長期的、穩(wěn)定的低毒性基因調(diào)控系統(tǒng)。隨后,我們構(gòu)建了基于配體作用的HDV核酶開關(guān)調(diào)節(jié)系統(tǒng),通過響應(yīng)茶堿分子濃度變化調(diào)節(jié)剪切,精確釋放靶基因的pri-miRNA,實(shí)現(xiàn)對腫瘤細(xì)胞抗凋亡基因Bcl-2的RNA干擾作用的實(shí)時(shí)有效調(diào)控,為今后RNA干擾在腫瘤細(xì)胞中的基因治療更安全有效的運(yùn)用提供理論基礎(chǔ)與研究方法。此外,本研究通過核酶開關(guān)的構(gòu)建,在真核動物細(xì)胞中建立了硫胺素焦磷酸(TPP)濃度響應(yīng)的EGFP熒光傳感器,可將其濃度的變化轉(zhuǎn)化為報(bào)告基因表達(dá)的改變,發(fā)展出對哺乳活細(xì)胞內(nèi)代謝物或因子的無標(biāo)記、無損傷、可視、高效的檢測方法。
[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.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q78

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