摘要
目的研究2型重组腺相关病毒(rAAV-2)介导的人凝血因子Ⅸ(hFⅨ)基因在脐血CD34+细胞及其子代细胞中的表达。方法采用rAAV-2/hFⅨ转导经预刺激的人脐血CD34+细胞,分别向粒单系、巨核系和红系分化培养21d,从转录水平、蛋白质水平和其功能活性检测hFⅨ的表达,同时检测子代细胞的活力、增殖倍数、各系标志分化抗原的表达及集落产率来评估rAAV-2对其增殖分化能力的影响。结果经测序证实转导组子代细胞的RNA可扩增出hFⅨcDNA片段,上清液中可检测到hFⅨ抗原的表达,每24h分泌量达14.10ng/106细胞。转导组和未转导组细胞培养21d后其活力、增殖倍数、标志性分化抗原的阳性率及集落产率差异均无统计学意义(P值均>0.05)。结论rAAV-2/hFⅨ能有效地转导人脐血CD34+细胞并在其子代细胞中表达具有凝血活性的hFⅨ,且对其体外培养21d的细胞增殖分化能力无明显影响。
Objective To investigate the expression of huamn coagulation factor Ⅸ （hFⅨ） gene in human umbilical cord blood （CB） CD34^＋ cells which was transfected with recombinant adeno-associated virus 2 （rAAV-2）. Method The CD34^＋ cells were transfected with rAAV-2/hFⅨ and cultured for 21. days for inducing differentiation into myeloid, erythroid and megakarocytie cells, respectively. The expression of hFIX was studied at mRNA, protein and biological activity levels. The cytotoxicity of rAAV-2 to CD34^＋ cells was evaluated by cell proliferation, cell vitality, CD antigen expressions and CFU yields. Results The hFⅨ mRNA was expressed in the cultured cells which was verified by RT-PCR and DNA sequencing. An elevated level of hFIX expression and biological activities were detected with a maximum amount of 14. 10 ng/10^6 cell · 24 h. During the period of 21 clay culture, the cell vitality, cell proliferation, CD antigen expression and CFU yields between the transfected and untransfected groups had no difference（ P 〉 0.05 ）. Conclusion The human CB CD34^＋ cells are able to produce functional hFIX after transducted by rAAV-2/hFⅨ. The cell proliferation and differentiation capacities of the host CD34^＋ cells were not affected by rAAV-2.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2005年第9期529-533,共5页
Chinese Journal of Hematology
基金
国家863计划基金资助项目(2001AA217181)
湖南省生命科学重点基金资助项目(99-02)
