摘要
近年来,基因治疗作为一种新的治疗方法给血友病A的治疗提供了新的思路。本研究旨在探讨在体外和NOD/SCID小鼠中应用慢病毒载体介导的血友病A基因疗法的可能性。构建含有B区缺失的人凝血因子Ⅷ(BDDhFⅧ)基因和IRES-eGFP编码序列的慢病毒表达载体pXZ9/BDDFⅧ。通过3质粒共转染293FT包装细胞,包装后感染293FT,HLF,Chang-liver和人骨髓间充质干细胞。在感染后分别通过酶联免疫吸附试验(ELISA),一期法,逆转录-聚合酶链反应(RT-PCR)和聚合酶链反应(PCR)法检测凝血因子Ⅷ(FⅧ)活性,FⅧ抗原,FⅧ的mRNA转录和基因整合情况。超速离心收集病毒颗粒,并通过门静脉注射感染NOD/SCID小鼠。ELISA分析小鼠血浆FⅧ抗原,荧光显微镜观察绿色荧光蛋白的表达,转导后1个月RT-PCR分析小鼠肝脏人FⅧ的转录情况。结果表明:成功制备高浓度的重组慢病毒,并能在体外高效转导靶细胞。感染后72 h能检测到高水平的FⅧ活性和FⅧ抗原。RT-PCR和PCR法能敏感检测到人FⅧ基因转录和整合至感染后的细胞中。在所有接受重组慢病毒颗粒注射后的NOD/SCID小鼠肝脏中均能检测到人FⅧ基因的转录,同时重组慢病毒也能在体内高效转导小鼠肝细胞。在感染后72 h小鼠血浆中人FⅧ水平为(49±6)mU,1周后为(54±8)mU,1个月后为(23±4)mU。结论:携带BDDhFⅧ基因的慢病毒颗粒在体内外能高效转导靶细胞,且所有被转导的靶细胞都能有效的分泌人FⅧ。经过门静脉注射慢病毒颗粒的NOD/SCID小鼠可以持续表达人FⅧ。
Recently,gene therapy has been become a promising approach to cure hemophilia A,a most common recessive bleeding disease.The aim of this study was to determine the perspect of lentiviral vector in hemophilia A gene therapy in vitro and in NOD/SCID mice.Lentivirus transfer vector pXZ9/BDDFⅧ containing human B-domain-deleted Factor Ⅷ-IRES-eGFP coding sequence and mock control pXZ9 were constructed.Lentivirus was prepared by co-transfecting 3 plasmids into 293FT cells.293FT,HLF,human bone marrow mesenchymal stem cells and Chang-liver cells were transfected with the prepared virus.Coagulant activity of human FⅧ,human FⅧ antigen,human FⅧ mRNA transcription and genomic integration were assayed by ELISA,one-step method,RT-PCR and PCR after infection.Lentiviral particles were concentrated by ultracentrifugation and NOD/SCID mice were transfected via portal vein injection.Human FⅧ antigen in mouse blood plasma was analyzed by ELISA.eGFP expression was observed by fluorescent microscopy and human FⅧ transcription in mouse liver was analyzed by RT-PCR at one month after transduction.The results showed that the high titer of recombinant virus was prepared and used to efficiently transduct the target cells in vitro.At 72 h after transfection,high levels of FⅧ activity and FⅧ antigen were detected.Human FⅧ gene transcription could be detected in the liver of NOD/SCID mice received lentiviral particles carrying FⅧ gene.Mouse hepatocytes were transfected with recombinant lentivirus efficiently in vivo.Human FⅧ level in mouse blood plasma reached to（49±6） mU,（54±8） mU and（23±4） mU at 72 h,one week and one month after transfection respectively.It is concluded that the lentiviral particles carrying BDDhFⅧ gene can high efficiently transfect the target cells both in vitro and in vivo,and the transfected target cells can secrete hFⅧ efficiently.The sustained expression of human FⅧ in NOD/SCID mice is observed after lentivirus transfection via portal vein injection.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2012年第3期658-663,共6页
Journal of Experimental Hematology
