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Expression of Human Clotting Factor IX Gene in Human Umbilical Cord Mesenchymal Stem Cells Mediated by Lentiviral Vector in Vitro

Author: LiWen
Tutor: ChenZuo
School: Chongqing Medical University
Course: Clinical hematology
Keywords: Human umbilical cord mesenchymal stem cells Human clotting factor IX Lentiviral vectortransfection
CLC: R554.1
Type: Master's thesis
Year: 2013
Downloads: 6
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Abstract


Objective To construct a lentiviral vector carrying human clottingfactor IX (hFIX) and to investigate its expression in human umbilical cordmesenchymal stem cells(hUCMSCs).Methods Human clotting factor IX gene fragment was obtained bypolymerase chain reaction (PCR) and cloned into lentiviral vector GV240to establish the expression vector GV240-hFIX-EGFP. Recomninantvector was identified by PCR and sequencing, then transfected into293T cells with liposome transfection to produce lentiviral particles. Theviral titer was determined by real-time quantitative PCR. hUCMSCs wereinfected at different multiple of infection (MOI).The transfectionefficiency is examined by Fluorescence microscopy and flow cytometry(FCM).The experiment is divided into three groups:experimental group,mock group and blank group, hUCMSCs were infected by recombinantlentivirus at the best MOI. The expression of hFIX was detected byRT-PCR and ELISA. The biological activity of hFIX was determined by one stage assay.Results Lentiviral vector system which produced high titerlentivirus at titers of up to2×108TU/mL was constructed successfully.When MOI was10, the transfection rate of hUCMSCs was92.92%. HFIXtranscription was detected by RT-PCR from infected cells. Aftertransfection, hUCMSCs expressed hFIX efficiently. Detections confirmedthat the activity of hFIX was (44.3±3.2)%and the concentration was(87.18±0.86) ng/(106·24h) at5days after infection. The expression couldmaintain at high level even29d after the infection.Conclusion The genetically modified hUCMSCs can expressbiologically active hFIX and thus serve as an efficient drug deliveryvehicle carrying hFIX used as a way of somatic gene therapy forhemophilia B.

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CLC: > Medicine, health > Internal Medicine > Blood and lymphatic system diseases > Blood diseases > Hemorrhagic disease > Hemophilia
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