重组腺相关病毒对造血干细胞高效转导的优化
发布时间：2018-07-13 08:26
【摘要】：目的：重组腺相关病毒(rAAV)被认为是最具应用前景的基因治疗病毒载体,应用其进行β-地中海贫血治疗是一项具有极大的应用价值的研究。然而rAAV感染效率低下一直是困扰研究者的难题,特别是对本身就难以感染的造血干细胞来说,其感染效率更加不理想。本研究从rAAV的包装到感染各过程进行研究探索,对比优化其包装及感染过程中的材料方法,并进行改进。致力于获得高效感染造血干细胞的AAV病毒及感染条件。 方法与结果：本研究主要集中于病毒包装过程和感染过程的优化与改进。在病毒的包装中,本实验分别采用PEI转染法及磷酸钙转染法包装病毒；超速离心层析纯化法及肝素-琼脂糖亲和纯化法纯化rAAV病毒,qPCR法及斑点杂交法进行病毒滴度测定,对比各种方法对rAAV感染活性的影响及区别,对rAAV病毒包装过程造成rAAV活性降低的步骤予以优化及改进,获得了一套完整的优化的包装高活性高感染效率的rAAV包装体系。在感染过程中的优化阶段,引入了自身互补腺相关病毒(scrAAV),同时结合采用rAAV外壳突变技术,对rAAV衣壳蛋白上暴露的酪氨酸位点进行突变,获得外壳突变型rAAV,很大的提高了rAAV对造血干细胞的感染效率。与此同时,在感染的过程中,发现感染体系中的各个感染条件对于rAAV的感染效率有十分大的影响。于是对感染条件：感染时间、血清浓度、感染滴度及感染后处理等进行了详细的探索,发现他们之间存在着复杂的相互影响,经过一系列实验,优化得到一个针对于造血干细胞的感染体系,相对以前的感染体系,大幅提高了感染效率。同时对感染前处理进行了探索,并首次发现ZY可以提高rAAV的感染效率,特别是结合羟基脲,可以很大幅度增加rAAV的感染效率,这是首次发现。 结论：通过对一系列的指标的优化和改进,获得了一套完整的优化的包装高活性高感染效率的rAAV包装体系。同时得到了针对造血干细胞的高效感染体系。于世界首次发现了ZY对rAAV感染效率的提高作用。
[Abstract]:Objective: recombinant adeno-associated virus (rAAV) is considered to be the most promising gene therapy vector for 尾 -thalassemia. However, the low infection efficiency of rAAV has been a difficult problem for researchers, especially for hematopoietic stem cells which are difficult to infect. In this study, the packaging of rAAV and the process of infection were studied and explored, and the methods of packaging and infection materials were optimized and improved. Aim to obtain highly efficient AAV virus infected with hematopoietic stem cells and infection conditions. Methods and results: this study focused on the optimization and improvement of virus packaging and infection process. The virus titers were detected by PEI transfection and calcium phosphate transfection respectively, and by ultracentrifugation chromatography purification, heparin-agarose affinity purification, rAAV virus titer detection by qPCR and dot blot hybridization. The effects and differences of various methods on rAAV infection activity were compared. The steps of rAAV activity reduction during rAAV packaging process were optimized and improved. A complete optimized rAAV packaging system with high activity and high infection efficiency was obtained. In the optimal stage of infection, self-complementary adeno-associated virus (scrAAV) was introduced, and the tyrosine site exposed to rAAV capsid protein was mutated by rAAV shell mutation technique. The rAAV infection efficiency of hematopoietic stem cells was greatly improved by obtaining rAAV. At the same time, in the process of infection, it was found that each infection condition in the infection system had a great influence on the infection efficiency of rAAV. So the infection conditions: time of infection, serum concentration, titer of infection and post-infection treatment were explored in detail. It was found that there was a complex interaction between them, and after a series of experiments, To optimize an infection system for hematopoietic stem cells, compared with the previous infection system, significantly improved the infection efficiency. It is the first time that ZY can improve the infection efficiency of rAAV, especially the combination of hydroxyurea, which can greatly increase the infection efficiency of rAAV. Conclusion: through the optimization and improvement of a series of indexes, a complete set of rAAV packaging system with high activity and high infection efficiency was obtained. At the same time, an efficient infection system for hematopoietic stem cells was obtained. The effect of ZY on the efficiency of rAAV infection was first found in the world.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2118769
[Abstract]:Objective: recombinant adeno-associated virus (rAAV) is considered to be the most promising gene therapy vector for 尾 -thalassemia. However, the low infection efficiency of rAAV has been a difficult problem for researchers, especially for hematopoietic stem cells which are difficult to infect. In this study, the packaging of rAAV and the process of infection were studied and explored, and the methods of packaging and infection materials were optimized and improved. Aim to obtain highly efficient AAV virus infected with hematopoietic stem cells and infection conditions. Methods and results: this study focused on the optimization and improvement of virus packaging and infection process. The virus titers were detected by PEI transfection and calcium phosphate transfection respectively, and by ultracentrifugation chromatography purification, heparin-agarose affinity purification, rAAV virus titer detection by qPCR and dot blot hybridization. The effects and differences of various methods on rAAV infection activity were compared. The steps of rAAV activity reduction during rAAV packaging process were optimized and improved. A complete optimized rAAV packaging system with high activity and high infection efficiency was obtained. In the optimal stage of infection, self-complementary adeno-associated virus (scrAAV) was introduced, and the tyrosine site exposed to rAAV capsid protein was mutated by rAAV shell mutation technique. The rAAV infection efficiency of hematopoietic stem cells was greatly improved by obtaining rAAV. At the same time, in the process of infection, it was found that each infection condition in the infection system had a great influence on the infection efficiency of rAAV. So the infection conditions: time of infection, serum concentration, titer of infection and post-infection treatment were explored in detail. It was found that there was a complex interaction between them, and after a series of experiments, To optimize an infection system for hematopoietic stem cells, compared with the previous infection system, significantly improved the infection efficiency. It is the first time that ZY can improve the infection efficiency of rAAV, especially the combination of hydroxyurea, which can greatly increase the infection efficiency of rAAV. Conclusion: through the optimization and improvement of a series of indexes, a complete set of rAAV packaging system with high activity and high infection efficiency was obtained. At the same time, an efficient infection system for hematopoietic stem cells was obtained. The effect of ZY on the efficiency of rAAV infection was first found in the world.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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相关期刊论文 前2条
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2 谢秋玲;郑云程;廖美德;;昆虫细胞Sf9在四种生物反应器中的培养[J];暨南大学学报(自然科学与医学版);2009年01期
，本文编号：2118769
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