【摘要】：目的：重組腺相關(guān)病毒(rAAV)被認(rèn)為是最具應(yīng)用前景的基因治療病毒載體,應(yīng)用其進(jìn)行β-地中海貧血治療是一項(xiàng)具有極大的應(yīng)用價(jià)值的研究。然而rAAV感染效率低下一直是困擾研究者的難題,特別是對(duì)本身就難以感染的造血干細(xì)胞來說,其感染效率更加不理想。本研究從rAAV的包裝到感染各過程進(jìn)行研究探索,對(duì)比優(yōu)化其包裝及感染過程中的材料方法,并進(jìn)行改進(jìn)。致力于獲得高效感染造血干細(xì)胞的AAV病毒及感染條件。 方法與結(jié)果：本研究主要集中于病毒包裝過程和感染過程的優(yōu)化與改進(jìn)。在病毒的包裝中,本實(shí)驗(yàn)分別采用PEI轉(zhuǎn)染法及磷酸鈣轉(zhuǎn)染法包裝病毒；超速離心層析純化法及肝素-瓊脂糖親和純化法純化rAAV病毒,qPCR法及斑點(diǎn)雜交法進(jìn)行病毒滴度測(cè)定,對(duì)比各種方法對(duì)rAAV感染活性的影響及區(qū)別,對(duì)rAAV病毒包裝過程造成rAAV活性降低的步驟予以優(yōu)化及改進(jìn),獲得了一套完整的優(yōu)化的包裝高活性高感染效率的rAAV包裝體系。在感染過程中的優(yōu)化階段,引入了自身互補(bǔ)腺相關(guān)病毒(scrAAV),同時(shí)結(jié)合采用rAAV外殼突變技術(shù),對(duì)rAAV衣殼蛋白上暴露的酪氨酸位點(diǎn)進(jìn)行突變,獲得外殼突變型rAAV,很大的提高了rAAV對(duì)造血干細(xì)胞的感染效率。與此同時(shí),在感染的過程中,發(fā)現(xiàn)感染體系中的各個(gè)感染條件對(duì)于rAAV的感染效率有十分大的影響。于是對(duì)感染條件：感染時(shí)間、血清濃度、感染滴度及感染后處理等進(jìn)行了詳細(xì)的探索,發(fā)現(xiàn)他們之間存在著復(fù)雜的相互影響,經(jīng)過一系列實(shí)驗(yàn),優(yōu)化得到一個(gè)針對(duì)于造血干細(xì)胞的感染體系,相對(duì)以前的感染體系,大幅提高了感染效率。同時(shí)對(duì)感染前處理進(jìn)行了探索,并首次發(fā)現(xiàn)ZY可以提高rAAV的感染效率,特別是結(jié)合羥基脲,可以很大幅度增加rAAV的感染效率,這是首次發(fā)現(xiàn)。 結(jié)論：通過對(duì)一系列的指標(biāo)的優(yōu)化和改進(jìn),獲得了一套完整的優(yōu)化的包裝高活性高感染效率的rAAV包裝體系。同時(shí)得到了針對(duì)造血干細(xì)胞的高效感染體系。于世界首次發(fā)現(xiàn)了ZY對(duì)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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本文編號(hào)：2118769