【摘要】：目的：重組腺相關病毒(rAAV)被認為是最具應用前景的基因治療病毒載體,應用其進行β-地中海貧血治療是一項具有極大的應用價值的研究。然而rAAV感染效率低下一直是困擾研究者的難題,特別是對本身就難以感染的造血干細胞來說,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行研究探索,對比優(yōu)化其包裝及感染過程中的材料方法,并進行改進。致力于獲得高效感染造血干細胞的AAV病毒及感染條件。 方法與結果：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。在病毒的包裝中,本實驗分別采用PEI轉染法及磷酸鈣轉染法包裝病毒；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法及斑點雜交法進行病毒滴度測定,對比各種方法對rAAV感染活性的影響及區(qū)別,對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,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,引入了自身互補腺相關病毒(scrAAV),同時結合采用rAAV外殼突變技術,對rAAV衣殼蛋白上暴露的酪氨酸位點進行突變,獲得外殼突變型rAAV,很大的提高了rAAV對造血干細胞的感染效率。與此同時,在感染的過程中,發(fā)現感染體系中的各個感染條件對于rAAV的感染效率有十分大的影響。于是對感染條件：感染時間、血清濃度、感染滴度及感染后處理等進行了詳細的探索,發(fā)現他們之間存在著復雜的相互影響,經過一系列實驗,優(yōu)化得到一個針對于造血干細胞的感染體系,相對以前的感染體系,大幅提高了感染效率。同時對感染前處理進行了探索,并首次發(fā)現ZY可以提高rAAV的感染效率,特別是結合羥基脲,可以很大幅度增加rAAV的感染效率,這是首次發(fā)現。 結論：通過對一系列的指標的優(yōu)化和改進,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。同時得到了針對造血干細胞的高效感染體系。于世界首次發(fā)現了ZY對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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

【參考文獻】

相關期刊論文 前2條

1 孫小娟;王峰;譚孟群;盧光t;;2型腺相關病毒轉導人骨髓CD34~+細胞與間充質干細胞[J];中南大學學報(醫(yī)學版);2006年01期

2 謝秋玲;鄭云程;廖美德;;昆蟲細胞Sf9在四種生物反應器中的培養(yǎng)[J];暨南大學學報(自然科學與醫(yī)學版);2009年01期

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