转导JAK2基因可促进原始多能造血细胞在体外的长期扩增  

Promotion of in vitro long term expansion of primitive multipotential hematopoietic cells by transduced JAK2 gene

作　　者：赵声明[1] 常乃柏[1] 顾惜春[1] 

机构地区：[1]北京医院血液科,100730

出　　处：《中华医学杂志》2004年第11期949-953,共5页National Medical Journal of China

基　　金：北京市自然科学基金资助项目 ( 70 42 0 5 5 )

摘　　要：目的　探讨激活JAK2信号通路是否能够长期扩增调控造血干祖细胞并评价扩增细胞的定向分化潜能。方法　构建克隆 1个含有JAK2基因和二聚化化学诱导物 (AP2 0 187)结合位点所组成的逆转录病毒载体。AP2 0 187可以使JAK2二聚化激活细胞内信号传导。将该载体转入小鼠原始骨髓造血细胞 ,在无血清培养基中 ,将JAK2转基因的细胞分为 :①空白对照组 ,②AP2 0 187组 ,③细胞因子联合组 [干细胞因子 (SCF) +Flt3 配体 (Flt3 L) ],④AP2 0 187+SCF +Flt3 L组。对扩增的细胞进行免疫表型、定向分化、造血祖细胞集落分析及脾集落形成单位的研究。结果　只有AP2 0 187+SCF+Flt3 L组能够使JAK2转基因的骨髓细胞持续大量对数级增殖。约 8d时 ,细胞数量可以达到扩增前的 10 19倍。扩增的细胞经流式细胞仪检测为 :Sca1阳性细胞为 5 2 %～ 98% ,C kit阳性率为 5 6 %～6 9 % ,CD34阳性率 4 0 %～ 85 % ;而其他表型TER119阳性 0～ 2 0 % ,CD4 1阳性 5 %～ 36 % ,B2 2 0阳性12 %～ 4 6 % ,Gr1阳性 6 %～ 17% ,CD11b阳性 35 %～ 4 6 % ,CD3为阴性。扩增细胞在SCF/粒细胞 巨噬细胞集落刺激因子 (GM CSF) /白介素 (IL) 3条件下 ,可分化形成明显的粒细胞和巨噬细胞 ;在SCF/红细胞生成素 (EPO)条件下 ,可分化为红细胞 ;Objective To explore whether activation of the JAK2 signaling pathway can stimulate long term expansion and regulation of hematopoietic stem cells/multipotential hematopoietic progentior cells (HSC/MHPC), and evaluate their potential ability of committed differentiation. Methods 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 transductioon, Transduced cells were divided equally into four groups as follow: ①No drug group, ②AP2018 alone group, ③SCF+Flt3 Ligand group, ④AP20187+SCF+Flt3 Ligand group. The expanded cells were further analyzed by phenotype, committed differentiation, progenitor colony assay as well as colony forming unite spleen. Results 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%～98%, while 56%～69% of cells expressed c kit, 40%～85% expressed CD34, About 12%～46% expressed B220, 6%～17% expressed Gr1, 0%～20% expressed TER119, 5%～36% expressed CD41, 35%～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 lymphoidcolonies in methylcellulose colonies assay. Colonies forming unites Spleen were obtained when the expanded cells injected into lethally irradiated mice. Conclusion The JAK2 mediated transgenic hematopoietic cells could be expanded and regulated in l

关 键 词：转导JAK2基因 原始多能造血细胞 体外扩增 造血干细胞 基因扩增 定向分化 

分 类 号：R55[医药卫生—血液循环系统疾病]