本文關(guān)鍵詞：碳量子點介導(dǎo)的基因傳遞與體細胞神經(jīng)分化研究　出處：《江蘇大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 熒光碳量子點 基因載體 細胞重編程 神經(jīng)干細胞 神經(jīng)元

【摘要】：目前,細胞替代療法是修復(fù)受損或病變中樞神經(jīng)系統(tǒng)的有效手段,直接重編程技術(shù)能夠?qū)Ⅲw細胞直接轉(zhuǎn)換為神經(jīng)干細胞(iNSCs)或神經(jīng)元(i Ns),可作為替代療法的細胞來源,促進中樞神經(jīng)系統(tǒng)的功能恢復(fù)。然而,在現(xiàn)代細胞重編程研究中多采用病毒作為基因載體,存在著基因突變、致瘤等安全隱患,碳量子點(CDs)作為一種新興的碳納米材料,是一種尺寸不超過10 nm的球形碳納米顆粒。碳量子點除具備優(yōu)異的光學(xué)性能外,還擁有其他量子點無法媲美的高生物相容性和低細胞毒性,故在生物醫(yī)藥領(lǐng)域備受關(guān)注,可應(yīng)用于熒光探針、生物成像、生化檢測和藥物傳遞等。近兩年,逐漸有報道將碳量子點應(yīng)用于基因載體,但是關(guān)于轉(zhuǎn)運機制等仍有待進一步探究。本課題擬利用海藻酸鈉為原材料,水熱合成法制備一種CDs,并將它與DNA結(jié)合自組裝為納米粒,作為多功能基因轉(zhuǎn)導(dǎo)系統(tǒng)開展體細胞向神經(jīng)分化的研究。第一章綜述本章主要闡述了研究對象,即碳量子點(CDs)不同的合成方法、性質(zhì)及其在多領(lǐng)域的應(yīng)用,重點論述了不同制備方法的優(yōu)缺點和不同領(lǐng)域的應(yīng)用前景,為CDs作為基因載體的研究提供可行性參考;同時,還圍繞體細胞重編程為神經(jīng)干細胞或神經(jīng)元的發(fā)展作簡單介紹,包括不同細胞來源、不同重編程方法的特點,并對其未來發(fā)展趨勢和面臨問題進行展望。最后,提出本課題的選題依據(jù)及思路,為論文的后續(xù)工作奠定基礎(chǔ)。第二章碳量子點的制備與表征本章主要將海藻酸鈉和3%過氧化氫作為反應(yīng)物,通過水熱法制備得到熒光多糖碳點(CP-CDs)。利用TEM、Zeta電位及熒光光譜等對其表征發(fā)現(xiàn),本課題中制備的CP-CDs平均粒徑在7 nm左右,為類球形顆粒,水分散性良好,表面帶有正電荷,并且具有激發(fā)光波長依賴性,可在不同波長激發(fā)光的照射下發(fā)射藍色、綠色及紅色三種顏色的熒光,熒光穩(wěn)定性強;利用元素分析、XPS對原材料海藻酸鈉進行考察,發(fā)現(xiàn)海藻酸鈉中含有N雜質(zhì),β消除反應(yīng)證實其中含有以O(shè)-糖肽方式連接的糖蛋白。因此,我們推斷海藻酸鈉在反應(yīng)中既作為碳源,又能作為陽離子化試劑,對CP-CDs表面進行鈍化修飾,提高其熒光性能。第三章碳量子點作為基因載體的初步研究本章主要圍繞CP-CDs能否作為基因載體展開工作,瓊脂糖凝膠電泳結(jié)果表明當(dāng)CP-CDs與DNA質(zhì)量比為40:1時可有效結(jié)合DNA。在此基礎(chǔ)上,將CP-CDs與DNA自組裝成納米粒,考察納米粒的細胞毒性、對DNA的保護、基因轉(zhuǎn)染效率、細胞內(nèi)轉(zhuǎn)運及攝取機理等。實驗結(jié)果表明,CP-CDs/DNA納米粒對3T6細胞幾乎無毒性,可保護DNA不被外界的核酶降解,其轉(zhuǎn)染效率明顯高于PEI和市售試劑Lip2000;轉(zhuǎn)染后4個小時細胞核內(nèi)發(fā)現(xiàn)目的基因,但是CP-CDs未進核,而是將DNA釋放后自身留在核外;吞噬抑制實驗發(fā)現(xiàn)小窩蛋白介導(dǎo)的細胞內(nèi)吞和網(wǎng)格蛋白介導(dǎo)的內(nèi)吞作用是納米粒攜帶基因進入細胞的主要途徑。第四章碳量子點介導(dǎo)的體細胞重編程研究本章主要以碳量子點作為基因載體,分別與五組基因組合自組裝成納米粒,體外轉(zhuǎn)染MEF細胞,將其直接轉(zhuǎn)換為iNSCs,并定向分化為神經(jīng)元,考察碳量子首次作為載體在細胞重編程中的應(yīng)用潛力,并篩選最佳因子組合。結(jié)果表明,誘導(dǎo)后的MEF細胞不但形態(tài)上類似iNSCs,還可表達iNSCs特異性標志物—Nestin和Pax;誘導(dǎo)i NSCs向神經(jīng)元分化,發(fā)現(xiàn)iNs可表達MAP2、Tau和Tuj1三種神經(jīng)元標志物;同時,對各組實驗結(jié)果對比分析后,篩選出Ascl1+Brn2雙因子組合的轉(zhuǎn)染效率最高,為最佳因子組合。
[Abstract]:At present, cell replacement therapy is an effective method to repair the damaged or disease of the central nervous system, direct reprogramming technique can be directly converted into neural stem cells (iNSCs) cells or neurons (I, Ns) can be used as alternative sources of cells, promote the recovery of the function of the central nervous system. However, in the study of modern cells programming in the use of virus as a gene carrier, there is a gene mutation, tumor and other safety hazards, carbon quantum dots (CDs) as a new kind of carbon nano materials, carbon nano particles are spherical with a size of less than 10 nm. The carbon quantum dots has excellent optical properties, but also has other quantum no comparable high biocompatibility and low toxicity, so it has attracted much attention in the field of biology and medicine, can be used in fluorescence probe, biological imaging, biochemical detection and drug delivery etc.. In recent years, gradually have reported carbon quantum dots Applied to the gene vector, but on the transport mechanism still needs to be further explored. This project intends to use the sodium alginate as raw material, hydrothermal synthesis of a CDs, and it will be combined with DNA self-assembled nanoparticles, as development of multifunctional gene transduction system to cell differentiation by research in the first chapter of this chapter. Mainly expounds the research object, namely carbon quantum dots (CDs) different synthesis methods, properties and applications in many fields, mainly discusses the different preparation methods and the advantages and disadvantages of different areas of application, provide a reference for the study of CDs as a gene carrier; at the same time, introduce development also around the body cell reprogramming of neural stem cells or neurons, including cells of different origins, characteristics of different reprogramming methods, and the problems facing the future development trends and prospects. Finally, proposed this topic selection Based on the questions and ideas, to lay the foundation for the follow-up work of the thesis. The second chapter carbon quantum dots preparation and characterization of this chapter will be sodium alginate and 3% hydrogen peroxide as a reactant, have been prepared by hydrothermal fluorescent polysaccharide (CP-CDs) carbon. Use of TEM, Zeta potential and fluorescence spectra were used to find. In this project, CP-CDs prepared an average size of about 7 nm, were spherical particles with good dispersion, water surface, with a positive charge, and with the excitation wavelength dependence, can emit blue in different wavelength excitation light irradiation, fluorescent green and red three colors, with strong fluorescence stability; Elemental analysis, XPS inspection of raw materials of sodium alginate, sodium alginate was found to contain N impurities, beta elimination reaction confirmed containing glycoproteins with O- peptide connection. Therefore, we concluded that sodium alginate in the reaction as a carbon source, and Can be used as the cationic reagent, modification of CP-CDs surface passivation, improve their fluorescence properties. The third chapter carbon quantum dots as a preliminary study of gene carrier in this chapter is mainly based on CP-CDs can be used as gene carrier work, agarose gel electrophoresis results showed that when CP-CDs and DNA mass ratio of 40:1 can be effectively combined with DNA. on the basis of this, the CP-CDs and DNA self assemble into rice, the cytotoxicity of nanoparticles, the protection of DNA, gene transfection efficiency, intracellular transport and uptake mechanism. The experimental results show that CP-CDs/DNA nanoparticles on 3T6 cells almost non-toxic, can protect the DNA from degradation of ribozyme outside, the transfection efficiency was significantly higher than that of PEI and commercially available reagents Lip2000 gene; found 4 hours after transfection, CP-CDs cell nucleus, but not into the nucleus, but after the release of their stay in the DNA nucleus; phagocytic inhibition of the caveolin mediated Endocytosis mediated endocytosis and clathrin mediated nanoparticles is the main way to carry genes into cells. Somatic cell reprogramming the fourth chapter CQDS mediated this chapter mainly carbon quantum dots as gene carrier, respectively, and five groups of gene combination self-assembled into nanoparticles, and transfected into MEF cells in vitro and it directly into iNSCs, and differentiate into neurons, the effects of carbon for the first time as a potential application in quantum carrier in cell reprogramming, and select the optimum factor combination. The results show that the induced MEF cells not only morphologically similar to iNSCs, but also the expression of iNSCs specific markers Nestin and Pax into neurons; differentiation of I NSCs, found that iNs expression of MAP2, Tau and Tuj1 three kinds of neuronal markers; at the same time, the experiments results of the comparative analysis, selected Ascl1+Brn2 two factor combination of the highest transfection efficiency, the best factor group .

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R450

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