Zhao Sheng-Ming, Chang Nai-Bai, Gu Xi-Chun
Department of Hematology, Beijing Hospital, Beijing 100730, China.
Zhonghua Yi Xue Za Zhi. 2004 Jun 2;84(11):949-53.
To explore whether activation of the JAK2-signaling pathway can stimulate long-term expansion and regulation of hematopoietic stem cells/multipotential hematopoietic progenitor cells (HSC/MHPC), and evaluate their potential ability of committed differentiation.
A retrovirus vector (RV) which contains JAK2 gene and two binding sites for a chemical inducers of dimerization (AP20187) was constructed. JAK2 can be dimerized by adding AP20187. Female C57BL/6 mice were euthanized and marrow cells were harvested. The RV vector was then transduced into murine bone marrow cells. Following transduction, Transduced cells were divided equally into four groups as follow: (1) No drug group, (2) AP2018 alone group, (3) SCF + Flt3-Ligand group, (4) AP20187 + SCF + Flt3-Ligand group. The expanded cells were further analyzed by phenotype, committed differentiation, progenitor colony assay as well as colony forming unite-spleen.
Only the group of AP20187 combined SCF and Flt3-Ligand have a significant sustained outgrowth. The cells reached at 10(19) fold on the day 80. The phenotype of expanded cells were checked by flow cytometry at various time points after two months in vitro culture and we repeated them in five separate experiments. Sca-1 was consistently expressed at the levels in 52% approximately 98%, while 56% approximately 69% of cells expressed c-kit, 40% approximately 85% expressed CD34, About 12% approximately 46% expressed B220, 6% approximately 17% expressed Gr1, 0% approximately 20% expressed TER119, 5% approximately 36% expressed CD41, 35% approximately 46% expressed CD11b and none expressed CD3. The expanded cells could differentiate into granulocytes, macrophages, erythocytes and megakaryocytes under different cytokines combination. They were also capable of forming BFU-E, CFU-GM, CFU-Mix and IL-7 responsive B-lymphoid colonies in methylcellulose colonies assay. Colonies forming unites-Spleen were obtained when the expanded cells injected into lethally irradiated mice.
The JAK2-mediated transgenic hematopoietic cells could be expanded and regulated in long-term period, and they are capable of maintaining multipotential differentiation. This research is setting up a fundamental basement for cell therapy in the future.
探讨JAK2信号通路的激活是否能刺激造血干细胞/多能造血祖细胞（HSC/MHPC）的长期扩增和调控，并评估其定向分化的潜在能力。
构建一种逆转录病毒载体（RV），其包含JAK2基因和两个二聚体化学诱导剂（AP20187）的结合位点。通过添加AP20187可使JAK2二聚化。对雌性C57BL/6小鼠实施安乐死并采集骨髓细胞。然后将RV载体转导至小鼠骨髓细胞中。转导后，将转导细胞平均分为四组：（1）无药物组，（2）单独AP2018组，（3）干细胞因子（SCF）+Flt3配体组，（4）AP20187+SCF+Flt3配体组。通过表型、定向分化、祖细胞集落测定以及脾集落形成单位对扩增细胞进行进一步分析。
只有AP20187联合SCF和Flt3配体组有显著的持续增殖。在第80天时细胞扩增至10^19倍。在体外培养两个月后的不同时间点，通过流式细胞术检测扩增细胞的表型，并在五个独立实验中重复检测。Sca-1持续表达水平约为52%至98%，而约56%至69%的细胞表达c-kit，约40%至85%表达CD34，约12%至46%表达B220，6%至17%表达Gr1，0%至20%表达TER119，5%至36%表达CD41，35%至46%表达CD11b，无细胞表达CD3。在不同细胞因子组合下，扩增细胞可分化为粒细胞、巨噬细胞、红细胞和巨核细胞。在甲基纤维素集落测定中，它们还能够形成爆式红系集落形成单位（BFU-E）、粒-巨噬细胞集落形成单位（CFU-GM）、混合集落形成单位（CFU-Mix）以及白细胞介素-7反应性B淋巴细胞集落。将扩增细胞注射到经致死剂量照射的小鼠体内可获得脾集落形成单位。
JAK2介导的转基因造血细胞能够长期扩增和调控，并且能够维持多能分化。本研究为未来的细胞治疗奠定了基础。
