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High Efficacy Transduction Hybrid AAV Vector and Its Application in β-thalassemia Gene Therapy Research

Author: LiuZhenQing
Tutor: TanMengQun
School: Central South University
Course: Physiology
Keywords: hematopoietic stem/progenitor cells mesenchymal stem cells adeno-associated virus β-thalassemia gene theropy
CLC: R556.61
Type: Master's thesis
Year: 2011
Downloads: 10
Quote: 0
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Abstract


ObjectiveThe object of this study is to optimize the construction of packaging purification hybrid AAV vector, carring the marked GFP gene; using each restructuring hybrid AAV vector to infect the human hematopoietic stem/ progenitor cells, to screen high efficient hybrid AAV vector in hematopoietic stem/ progenitor cells transduction, so as to construct the type AAV vectorr containingβ-globin gene, and to transductβ-thalassemia aborted fetus’s bone marrow hematopoietic stem/progenitor cells. Then transfected cells were transplanted into illuminated BALB/c nude mice and to systematically check theβ-globin gene in rats’bodies. Finally to select high-effiecient hybrid rAAV vector.Methods and Results1. Hematopoietic stem/progenitor cells and mesenchymal stem cell separation and cultureIn this study we adopted density gradient centrifugation combined with immune magnetic beads separation method to obtain human hematopoietic stem/progenitor cells, using FCM method to detect separation purity and group culivation method to test differentiation potential and the biology activity, density gradient centrifugation combining stick wall training method to obtain human bone marrow MSCs in vitro, respectively inducing MSCs to separate into fat and osteoblast cells. Results showed that through the immune magnetic beads separation can gain high purity hematopoietic stem/progenitor cells. Colony culture illustrated it had relatively high differentiation potential and the biology activity; In vitro original generation training and subculture can obtain MSCs with high amplification ability.2. The obtaintion of adeno-associated virus vectorWe adopted calcium superphosphate coprecipitation, three plasmid cotransfection carrying heparin-agarose method to pack and purify rAAV virus vector; containing purpose gene. We used SDS-PAGE protein electrophoresis, dot blot hybridization and cells infection to detect rAAV purity, titer and biological activity. Results showed that the packing purified rAAV had high biology activity, which can successfully infect cells.3. Screening rAAV vector with high efficiency in transduction of hematopoietic stem/progenitor cellsUsing rAAV1-GFP, rAAV2-GFP, rAAV6-GFP respectively to transduction human hematopoietic stem/progenitor cells, FCM method to detect its infect efficiency. The results showed that rAAV1-GFP or rAAV6-GFP> rAAV2-GFP. Based on the results to packing, purification and to obtain rAAV1-β-globin、rAAV6-β-globin. We used BALB/c nude mice experiment for further screening rAAV vector with high efficiency in transfecting hematopoietic stem/ progenitor cells. After illumination with X ray, BALB/c nude mice were transplanted by rAAV-β-globin infectedβ- thalassemia aborted fetus’s bone marrow hematopoietic stem/progenitor cells. We check the expression of humanβ-globin in nude mice’s blood. Using Western Blot detection ofβ-globin protein in nude rats’peripheral blood and IPP gray scanning software to analyse the results, which turned out to be rAAV1 17.12, rAAV6 13.6.ConclusionThis study has successfully separated and cultured human hematopoietic stem/ progenitor cells and bone marrow MSCs in vitro,which possessed efficient augmentation and differentiated ability, and can effectively mediated the expression of GFP in hematopoietic stem/ progenitor cells and MSCs through rAAV vector. In addition, we selected high transduction human hematopoietic stem/ progenitor cells rAAV vector rAAV1 and rAAV6. Furthermore through in vivo study, we found that the transduction efficient of rAAV1 was higher than rAAV6.

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CLC: > Medicine, health > Internal Medicine > Blood and lymphatic system diseases > Blood diseases > Anemia > Hemolytic anemia > Congenital hemolytic anemia
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