碳量子点介导的基因传递与体细胞神经分化研究

发布时间：2018-01-04 07:29

本文关键词：碳量子点介导的基因传递与体细胞神经分化研究　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：目前,细胞替代疗法是修复受损或病变中枢神经系统的有效手段,直接重编程技术能够将体细胞直接转换为神经干细胞(iNSCs)或神经元(i Ns),可作为替代疗法的细胞来源,促进中枢神经系统的功能恢复。然而,在现代细胞重编程研究中多采用病毒作为基因载体,存在着基因突变、致瘤等安全隐患,碳量子点(CDs)作为一种新兴的碳纳米材料,是一种尺寸不超过10 nm的球形碳纳米颗粒。碳量子点除具备优异的光学性能外,还拥有其他量子点无法媲美的高生物相容性和低细胞毒性,故在生物医药领域备受关注,可应用于荧光探针、生物成像、生化检测和药物传递等。近两年,逐渐有报道将碳量子点应用于基因载体,但是关于转运机制等仍有待进一步探究。本课题拟利用海藻酸钠为原材料,水热合成法制备一种CDs,并将它与DNA结合自组装为纳米粒,作为多功能基因转导系统开展体细胞向神经分化的研究。第一章综述本章主要阐述了研究对象,即碳量子点(CDs)不同的合成方法、性质及其在多领域的应用,重点论述了不同制备方法的优缺点和不同领域的应用前景,为CDs作为基因载体的研究提供可行性参考;同时,还围绕体细胞重编程为神经干细胞或神经元的发展作简单介绍,包括不同细胞来源、不同重编程方法的特点,并对其未来发展趋势和面临问题进行展望。最后,提出本课题的选题依据及思路,为论文的后续工作奠定基础。第二章碳量子点的制备与表征本章主要将海藻酸钠和3%过氧化氢作为反应物,通过水热法制备得到荧光多糖碳点(CP-CDs)。利用TEM、Zeta电位及荧光光谱等对其表征发现,本课题中制备的CP-CDs平均粒径在7 nm左右,为类球形颗粒,水分散性良好,表面带有正电荷,并且具有激发光波长依赖性,可在不同波长激发光的照射下发射蓝色、绿色及红色三种颜色的荧光,荧光稳定性强;利用元素分析、XPS对原材料海藻酸钠进行考察,发现海藻酸钠中含有N杂质,β消除反应证实其中含有以O-糖肽方式连接的糖蛋白。因此,我们推断海藻酸钠在反应中既作为碳源,又能作为阳离子化试剂,对CP-CDs表面进行钝化修饰,提高其荧光性能。第三章碳量子点作为基因载体的初步研究本章主要围绕CP-CDs能否作为基因载体展开工作,琼脂糖凝胶电泳结果表明当CP-CDs与DNA质量比为40:1时可有效结合DNA。在此基础上,将CP-CDs与DNA自组装成纳米粒,考察纳米粒的细胞毒性、对DNA的保护、基因转染效率、细胞内转运及摄取机理等。实验结果表明,CP-CDs/DNA纳米粒对3T6细胞几乎无毒性,可保护DNA不被外界的核酶降解,其转染效率明显高于PEI和市售试剂Lip2000;转染后4个小时细胞核内发现目的基因,但是CP-CDs未进核,而是将DNA释放后自身留在核外;吞噬抑制实验发现小窝蛋白介导的细胞内吞和网格蛋白介导的内吞作用是纳米粒携带基因进入细胞的主要途径。第四章碳量子点介导的体细胞重编程研究本章主要以碳量子点作为基因载体,分别与五组基因组合自组装成纳米粒,体外转染MEF细胞,将其直接转换为iNSCs,并定向分化为神经元,考察碳量子首次作为载体在细胞重编程中的应用潜力,并筛选最佳因子组合。结果表明,诱导后的MEF细胞不但形态上类似iNSCs,还可表达iNSCs特异性标志物—Nestin和Pax;诱导i NSCs向神经元分化,发现iNs可表达MAP2、Tau和Tuj1三种神经元标志物;同时,对各组实验结果对比分析后,筛选出Ascl1+Brn2双因子组合的转染效率最高,为最佳因子组合。
[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 .

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R450

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