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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 human coagulation factor Ⅸ (hFⅨ) gene in cord blood CD34 + cells and its progeny cells mediated by recombinant adeno-associated virus type 2 (rAAV-2). Methods Human umbilical cord blood CD34 + cells pre-stimulated with rAAV-2 / hFⅨ were transduced to differentiate into monolayer, megakaryocyte and erythroid cells for 21 days respectively. The expression of hFⅨ was detected by transcriptional level, protein level and functional activity The effect of rAAV-2 on the proliferation and differentiation of progeny cells was evaluated by measuring the viability and multiplication of progeny cells, the expression of differentiated antigens in each line and the yield of colony. Results The sequence of hFIX cDNA was amplified by RNA from the progeny of transduction group. The expression of hFIX antigen was detected in the supernatant, and the secretion of hFIX was 14.10 ng / 106 cells per 24h. After 21 days of culture, the viability, multiplication ratio, positive rate of labeled differentiated antigen and colony yield did not show significant difference (both P values> 0.05). Conclusion rAAV-2 / hFⅨ can effectively transduce human umbilical cord blood CD34 + cells and express clotting-active hFⅨ in its progeny cells, and has no significant effect on the proliferation and differentiation of cells cultured for 21 days.