JAK2转基因骨髓造血干/祖细胞体外长期扩增调控及定向分化的研究

Long term regulated expansion and committed differentiation of JAK2 gene transfected hematopoietic stem/progenitor cells in vitro

目的 探讨酪氨酸激酶JAK2转基因骨髓造血干 祖细胞体外长期扩增调控和定向分化潜能的可行性。方法 克隆了一个MSCV based逆转录病毒载体称作MGI F2Jak2 ,它编码一个绿色荧光蛋白 (GFP)和两个改进的FK5 0 6结合蛋白 (F36v)与JAK2组成的融合蛋白 ,F36v是二聚化化学诱导物AP2 0 187的结合位点。应用该载体转染GpE +86包装细胞株 ,将C5 7BL 6小鼠的骨髓细胞与照射过15 0 0cGy的GpE +86产毒细胞株共同培养实现基因转导。转导后的细胞在X VIVO 15培养体系中扩增 ,分为①空白对照组 ;②AP2 0 187组 ;③干细胞因子 (SCF)组 ;④AP2 0 187+SCF组。扩增的细胞用直接法标记藻红蛋白荧光素 (PE)结合的抗Sca1、c kit、CD34 、Gr1、CD1 1b、TER 119、CD4 1 、B2 2 0和CD3单克隆抗体以流式细胞仪检测 ,并进行定向分化、祖细胞集落培养和脾细胞集落形成单位 (CFU S)的研究。结果 只有AP2 0 187+SCF组可以持续地使转基因的骨髓造血干 祖细胞大量增殖 ,扩增 80d后细胞可达10 1 4倍 ,细胞倍增时间约 30h ,扩增细胞的表型为CD34 、c kit和Sca1等干细胞表面标志强阳性 ,其它细胞表面标志如Gr1、CD1 1b、TER119、CD4 1 、B2 2 0、CD3接近阴性。所扩增的细胞在不同的细胞因子组合下 ,可定向分化为粒细胞、巨噬细胞、红细胞、

Objective To explore the feasibility of regulated expansion and committed differentiation potential of JAK2 gene modified hematopoietic stem/progenitor cells in vitro.Methods A murine stem cell virus (MSCV) based retroviral vector MGI-F2Jak2, which encodes a green fluorescent protein (GFP) and a fusion protein containing two copies of modified FK506 binding protein (F36v) linked tyrosine kinase JAK2 was cloned. F36v served as a high-affinity binding site for dimerizer AP20187. GpE+86 packaging cell was transfected with this vector. Bone marrow cells from C57BL/6 mice were transduced by co-cultured with irradiated (1500 cGy) GpE+86 producer clone for 48 h. Transduced marrow cells were expanded in X-VIVO 15 and divided into four groups as follows:①control group;② AP20187 alone group;③SCF alone group and ④ AP20187+SCF group. The phenotypes of the expanded cells were analyzed by directly phycoerythrin-labeled anti-Sca1, c-kit, CD 34, Gr1, CD 11b, TER119, CD 41, B220 and CD 3 monoclonal antibodies for flow cytometry. Committed differentiation, progenitor colony assay and spleen colony forming units (CFU-S) were further evaluated. Results A significant sustained outgrowth of transduced marrow cells was obtained only in the AP20187+SCF group. Cells expanded up to 10 14-fold after 80 days culture. The doubling time was about 30 hs. The phenotypes of the expanded cells were homogeneously strong positive for CD 34, c-kit and Sca1, while were almost negative for Gr1, CD 11b, TER119, CD 41, B220 and CD 3. Functional assay demonstrated that the expanded cells had multipotential to differentiate into granulocyte, macrophage, erythrocyte, or B-cells under different cytokines combinations. A prominent megakaryocytic differentiation was observed when cultured with SCF/Tpo/IL-11 combination. The expanded cells were also capable of forming BFU-E,CFU-GM and CFU-Mix in methylcellulose colony assay. The expanded cells over three months could still form CFU-S. Conclusions AP20187 combined SCF mediated activation of JAK2 signaling domain can dramatically expand hematopoietic stem/progenitor cells, and the expanded cells can be regulated and committed to differentiate into multilineage cells. This system may provide important insights into stem cell biology and may be promising for gene and cell therapy.