【摘要】：隨著納米科技的快速發(fā)展,采用納米技術(shù)制備的納米材料成為了當前的研究熱點。納米材料由于具有特殊的物理化學性質(zhì),已經(jīng)被廣泛應(yīng)用于生物醫(yī)學領(lǐng)域,并取得了很多有價值的應(yīng)用成果。聚合酶鏈式反應(yīng)(Polymerase Chain Reaction,PCR技術(shù))是一種基于DNA擴增的分子生物學技術(shù),也是臨床醫(yī)學中的檢測疾病的重要手段,但是PCR擴增中時常出現(xiàn)的低效率和非特異性問題限制了該技術(shù)的廣泛應(yīng)用�！凹{米PCR”概念的引進為PCR技術(shù)的完善提供了一種獨特的思路。此外,基因治療(Gene therapy)作為一種新型的治療疾病的方法被認為是藥學和醫(yī)學領(lǐng)域的一次革命。與傳統(tǒng)治療方法相比,基因治療有望從根本上治療疾病,已成為目前國際學術(shù)界研究的焦點。然而,基因傳遞中使用的基因傳遞載體經(jīng)常具有很高的毒性或者較低的轉(zhuǎn)染效率,以納米材料為代表的非病毒載體逐漸成為科學家們研究的“新寵”。本碩士論文主要是以超支化聚乙烯亞胺(Polyethylenimine,PEI)包裹的納米金顆粒作為優(yōu)化劑應(yīng)用于PCR技術(shù),并探討其優(yōu)化機理,同時作為一種負載外源正�；虻膫鬟f載體應(yīng)用于基因治療的研究。我們希望通過本論文的研究,開發(fā)出一種既可以優(yōu)化PCR技術(shù)又能作為理想的基因傳遞載體的多功能性的納米材料。在第二章中,首先我們通過選擇價廉易得的PEI作為樹狀大分子的替代品,在PEI表面修飾聚乙二醇(mPEG),用來提高納米材料的穩(wěn)定性和生物相容性。并以此為模板,利用硼氫化鈉還原法,調(diào)控氯金酸與PEI的投料摩爾比,從而調(diào)控納米材料的結(jié)構(gòu),表面電勢和粒徑大小,最終合成了一系列包裹不同數(shù)量納米金顆粒的PEG-Au PENPs(PEGylated PEI-entrapped Entrapped Gold Nanoparticles)。另外,通過表征數(shù)據(jù)可知:所合成的PEG-Au PENPs粒徑較小、分布均勻、具有良好的生物相容性和較高的熱導(dǎo)率。在第三章中,根據(jù)相關(guān)文獻,我們首先建立了兩種PCR擴增體系:二輪易錯PCR反應(yīng)體系和高GC含量(74%GC含量)PCR反應(yīng)體系。然后以這兩種體系作為PCR優(yōu)化劑的篩選平臺,通過一系列優(yōu)化實驗和凝膠電泳實驗來評估PEG-Au PENPs的優(yōu)化能力。此外,我們通過系統(tǒng)的實驗設(shè)計,進一步探討了納米材料對PCR技術(shù)的優(yōu)化機理。結(jié)果表明:所有的PEG-Au PENPs都可以提高PCR反應(yīng)的效率和特異性。值得指出的是,我們發(fā)現(xiàn)納米材料在不同的PCR體系中,可能展示著不同的優(yōu)化機理。在二輪易錯PCR體系中,PEG-Au PENPs的優(yōu)化作用可能是由于其表面電荷介導(dǎo)的靜電作用所導(dǎo)致的;而在高GC含量PCR體系中,PEG-Au PENPs的良好的熱傳遞性能可能是優(yōu)化PCR反應(yīng)的主要原因。在第四章中,我們首先通過凝膠阻滯實驗測試了PEG-Au PENPs壓縮pDNA的能力,然后通過增強型綠色熒光蛋白(EGFP)基因和熒光素酶(Luc)基因的表達實驗來衡量材料的轉(zhuǎn)染效率。最后,通過流式細胞術(shù)以及材料/pDNA復(fù)合物在細胞內(nèi)的定位實驗進一步研究了材料的轉(zhuǎn)染機理。結(jié)合第二章的細胞毒性實驗,我們發(fā)現(xiàn):與PEI相比,PEG-Au PENPs不僅可以明顯的降低細胞毒性,而且能保持較高的轉(zhuǎn)染效率,另外,PEG-Au PENPs/pDNA復(fù)合物與PEI/pDNA復(fù)合物在細胞內(nèi)有著共同的胞內(nèi)傳遞途徑。
[Abstract]:With the rapid development of nanotechnology, nanomaterials prepared by nanotechnology have become the focus of current research. Nanomaterials have been widely used in biomedical fields due to their special physical and chemical properties. Many valuable applications have been achieved. Polymerase Chain Reaction (PCR) Technology The introduction of the concept of "nano-PCR" provides a unique way of thinking for the improvement of PCR technology. In addition, gene therapy Gene therapy, as a new method of treating diseases, is regarded as a revolution in pharmacy and medicine. Compared with traditional therapy, gene therapy is expected to treat diseases fundamentally, and has become the focus of international academic research. However, gene delivery vectors used in gene delivery are often highly toxic. Non-viral vectors, such as nano-materials, have gradually become the "new favorite" of scientists because of their low transfection efficiency.In this dissertation, gold nanoparticles encapsulated by Hyperbranched polyethylenimine (PEI) were used as optimizers in PCR, and their optimization mechanism was discussed as well as as as a load. We hope to develop a kind of multifunctional nano-material which can optimize PCR technology and can be used as an ideal gene delivery vector. In the second chapter, we first choose the cheap and easy-to-obtain PEI as a substitute for dendrimers. Polyethylene glycol (mPEG) was modified on the surface of PEI to improve the stability and biocompatibility of nano-materials. As a template, sodium borohydride reduction method was used to adjust the molar ratio of chloroauric acid to PEI, so as to control the structure, surface potential and particle size of nano-materials. Finally, a series of nano-gold particles were synthesized. PEG-Au PENPs (PEGylated PEI-entrapped Entrapped Gold Nanoparticles). In addition, the characterization data showed that the synthesized PEG-Au PENPs had small particle size, uniform distribution, good biocompatibility and high thermal conductivity. R reaction system and high GC content (74% GC) PCR reaction system were used as the screening platform for PCR optimizers. A series of optimization experiments and gel electrophoresis experiments were carried out to evaluate the optimization ability of PEG-Au PENPs. The results showed that all PEG-Au PENPs could improve the efficiency and specificity of PCR reaction. It is worth pointing out that nanomaterials may exhibit different optimization mechanisms in different PCR systems. In Chapter 4, we first tested the ability of PEG-Au PENPs to compress pDNA by gel blockade, and then confirmed the expression of enhanced green fluorescent protein (EGFP) and luciferase (Luc) genes. Finally, the transfection mechanism of PEG-Au PENPs was further studied by flow cytometry and the localization of material/pDNA complex in cells. Combined with the cytotoxicity experiment in Chapter 2, we found that PEG-Au PENPs not only significantly reduced the cytotoxicity of PEI, but also maintained a high level of transfection. In addition, PEG-Au PENPs/pDNA complex and PEI/pDNA complex share the same intracellular transmission pathway.
【學位授予單位】：東華大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R450

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