本文選題：基因載體 + 轉(zhuǎn)染效率��； 參考：《武漢理工大學》2015年碩士論文

【摘要】：基因治療已經(jīng)開始用于腫瘤、癌癥及各種遺傳性疾病的治療當中,臨床效果較傳統(tǒng)治療顯著,且應(yīng)用前景廣闊。而要實現(xiàn)高效的基因治療,設(shè)計高效低毒的基因載體是其中一個關(guān)鍵的環(huán)節(jié)�；蜉d體包括病毒類載體、非病毒類載體,相對于病毒載體,非病毒載體尤其是陽離子非病毒載體具有低細胞毒性、低免疫性、易合成等特點,已成為目前的研究熱點。殼聚糖作為天然的多糖,具有無毒,可降解,生物相容性好,且分子中含有大量的氨基,能與質(zhì)粒DNA復(fù)合形成納米粒,已有大量報道將其作為基因載體應(yīng)用于基因治療。但是殼聚糖水溶性差,與DNA復(fù)合緊密而難于在體內(nèi)釋放DNA,轉(zhuǎn)染效率較低,臨床應(yīng)用受到限制。因此,尋找適合的途徑得到新型陽離子殼聚糖衍生物來作為基因載體,提高它的轉(zhuǎn)染效率,是研究者努力的方向。本論文實驗第一部分利用邁克爾加成反應(yīng)合成了一系列新型水溶性殼聚糖衍生物。用核磁共振氫譜對載體材料進行化學表征,以確定其結(jié)構(gòu)。用凝膠電泳實驗觀察殼聚糖衍生物與質(zhì)粒DNA的結(jié)合能力,用電子掃描電鏡來觀察粒子的形狀大小,測定粒子粒徑及電動電勢,用MTT法檢測復(fù)合體的細胞毒性,通過體外細胞培養(yǎng)來測定殼聚糖衍生物的轉(zhuǎn)染效率。實驗結(jié)果表明,與殼聚糖相比,新型殼聚糖衍生物具有較低的細胞毒性,較好的DNA復(fù)合能力,在293 T細胞和HeLa細胞中有更高的轉(zhuǎn)染效率�；诙蜴I對不同濃度的谷胱甘肽濃度有特異響應(yīng)性,并且細胞中的谷胱甘肽濃度高于血液中的谷胱甘肽濃度,癌細胞中的谷胱甘肽濃度約為正常細胞內(nèi)的四倍,本論文實驗第二部分我們以透明質(zhì)酸為骨架,引入二硫鍵,后接枝四乙烯五胺制備出一種基于透明質(zhì)酸的陽離子高分子載體,用紅外、核磁共振氫譜(1H NMR)對載體材料進行結(jié)構(gòu)表征。通過與質(zhì)粒DNA形成復(fù)合物,并用瓊脂糖凝膠電泳分析其結(jié)合DNA的能力及在高濃度谷胱甘肽刺激下釋放DNA的能力;測定復(fù)合粒子的粒徑、電勢,并在掃描電子顯微鏡下觀察復(fù)合物的形態(tài);用MTT法檢測復(fù)合體的細胞毒性。結(jié)果表明,新合成的透明質(zhì)酸陽離子衍生物易與DNA復(fù)合,并在模擬細胞內(nèi)高濃度谷胱甘肽刺激下釋放DNA,且細胞毒性較低,是一種潛在的基因載體。設(shè)計開發(fā)新型的陽離子基因載體具有重要的意義。本論文通過對殼聚糖和透明質(zhì)酸的化學修飾,分別得到相應(yīng)的陽離子高分子衍生物,實驗結(jié)果顯示,所制備的新型陽離子高分子都具有較低的細胞毒性,較好的DNA復(fù)合能力,在作為基因載體方面具有潛在的應(yīng)用。
[Abstract]:Gene therapy has been used in the treatment of cancer, cancer and various hereditary diseases. To achieve efficient gene therapy, the design of efficient and low-toxic gene vector is one of the key links. Gene vectors include viral vectors, non-viral vectors. Compared with viral vectors, non-viral vectors, especially cationic non-viral vectors, have the characteristics of low cytotoxicity, low immunity and easy synthesis. Chitosan, as a natural polysaccharide, is nontoxic, biodegradable, biocompatible, and has a large amount of amino acids in the molecule, which can compound with plasmid DNA to form nanoparticles. It has been reported that chitosan can be used as gene vector in gene therapy. However, chitosan is difficult to release DNA in vivo because of its poor water solubility and compounding with DNA, so its transfection efficiency is low, and its clinical application is limited. Therefore, to find a suitable way to obtain new cationic chitosan derivatives as gene vector and improve its transfection efficiency is the direction of researchers' efforts. In the first part of this thesis, a series of new water-soluble chitosan derivatives were synthesized by Michael addition reaction. The support material was chemically characterized by nuclear magnetic resonance spectroscopy (NMR) to determine its structure. The binding ability of chitosan derivatives to plasmid DNA was observed by gel electrophoresis, the shape and size of particles, the particle size and electromotive force were measured by electron scanning electron microscope (SEM), and the cytotoxicity of the complex was detected by MTT method. The transfection efficiency of chitosan derivatives was determined by cell culture in vitro. The results showed that compared with chitosan, the new chitosan derivatives had lower cytotoxicity, better DNA recombination ability and higher transfection efficiency in 293T cells and HeLa cells. Based on the specific response of disulfide bond to different concentrations of glutathione, the concentration of glutathione in cells was higher than that in blood, and the concentration of glutathione in cancer cells was about four times as high as that in normal cells. In the second part of this thesis, we used hyaluronic acid as the skeleton, introduced disulfide bond, and then grafted tetraethylenepentylamine to prepare a kind of cationic polymer carrier based on hyaluronic acid. The structure of the carrier was characterized by 1H NMR. By forming a complex with plasmid DNA, using agarose gel electrophoresis to analyze its ability to bind DNA and release DNA under high concentration of glutathione, to determine the particle size and potential, The morphology of the complex was observed under scanning electron microscope (SEM), and the cytotoxicity of the complex was detected by MTT assay. The results showed that the newly synthesized hyaluronic acid cationic derivatives were easy to compound with DNA and released DNA under the stimulation of high concentration of glutathione in mimic cells, and the cytotoxicity was low, so it was a potential gene vector. It is of great significance to design and develop a new cationic gene vector. In this paper, the corresponding cationic polymer derivatives were obtained by chemical modification of chitosan and hyaluronic acid. The experimental results showed that the new cationic polymers had lower cytotoxicity and better DNA recombination ability. It has potential application as gene vector.
【學位授予單位】：武漢理工大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R450

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