靶基因调控的脐血干/祖细胞体外长期扩增与调控的实验研究

Targeted Expansion and Regulation of Genetically Modified Cord Blood Stem/Progenitor Cells in vitro

目的探讨转基因JAK2介导的脐血干祖细胞长期扩增调控的可行性和安全性。方法构建逆转录病毒载体MGI-F2JAK2,内含有JAK2基因的功能催化区和两个与小分子靶向基因合成药物(AP20187)结合的位点蛋白(2xF36v,F2)组成。AP20187可与F36v特异结合引起JAK2二聚化而激活细胞内信号传导。该载体同时含有绿色荧光蛋白(GFP)报告基因,用作检测细胞增殖的标记。应用MiniMACS免疫磁珠分选系统纯化分离脐血CD34+细胞,用含JAK2的逆转录病毒上清转染脐血CD34+细胞。转导后的CD34+细胞在SCF、FL、TPO、IL-6细胞因子的联合培养条件下,以不加或加入AP20187分别作为对照组和实验组。定期检测CD34+细胞基因转移后GFP动态变化、细胞免疫标记、造血祖细胞集落培养和裸鼠致瘤试验。结果分选的CD34+细胞纯度为91%以上,基因转移率为49．3%±6．2%；实验组AP20187+SCF+FL+TPO+IL-6(ASFTI)与对照组SCF+FL+TPO+IL-6(SFTI)组均可获得CD34+细胞大量扩增。随着培养时间的延长,实验组扩增的CD34+细胞GFP阳性率由基线水平逐渐上升于第11周时达到95%以上,而对照组GFP阳性率逐渐下降到基线水平以下并逐渐消失。在培养6周左右,实验组CD34+CD38-、CD34+CD38+细胞亚群扩增倍数分别为(228．26±32．31)、(321．48±40．52),分别与对照组相比,差异有显著性。ASFTI组转基因CD34+细胞于12周后仍可产生造血祖细胞集落(BFU-E、CFU-GM、CFU-Mix)。扩增后CD34+细胞检测染色体核型正常,裸鼠实验无致瘤特性。结论转染JAK2基因的人脐血CD34+细胞协同其他细胞因子可以体外长期扩增脐血干祖细胞,对今后开展干细胞治疗某些遗传性血液病有潜在的应用价值。

Objective To explore the feasibility of long-term regulated expansion of genetically modified human CD34＋ purified cord blood cells in vitro and evaluate their biological safety. Methods A retrovirus （RV） vector which contains JAK2 catalytic domain and two binding sites for a chemical inducers of dimerization （AP20187） was cloned. JAK2 can be dimerized by adding AP20187. The RV vector encoding a green fluorescent protein （GFP）. CD34 ＋ cells were isolated from cord blood cells by MiniMACS. The purified CD34＋cells were tranduced with supernatant from the RV packaging cell line. Following transduction, cells were cultured in suspension with in a combination of growth factors （SCF, Flt3, TPO and IL-6）, either in the presence or absence of AP20187 （100 nM）. We then further analyzed their GFP expression efficiency, immunophenotype and multipotent committed progenitor colony assay for JAK2 transduced CD34＋cells. Meanwhile, cells were evaluated the safety, karyotype and nude mice model tumorigenesis. Results The purity of selected CD34＋ cells was over 91% and gene transfer rate was 49.3%±6. 2%. We obtained CD34＋cells proliferation both in the presence of ASFTI and SFTI groups, but the GFP expression efficiency significantly differed between two groups. The percentage of GFP＋ cells over time in culture was strongly influenced by the presence of the AP20187. The absence of AP20187 group was associated with a steady decline in the percentage of GFP ＋ cells from baseline immediately following transduction, gradually to disappear in culture. In contrast, the addition of AP20187 group consistently produced a rise to the 95% peak level in the percentage of GFP＋ cells by the end of l lth week of culture. Thereafter, cultures performed in the presence of AP20187 maintained a higher frequency of GFP＋ cells than cultures performed in the absence of AP20187. ASFTI group appears to continue express CD34 ＋ CD38 /CD34 ＋ CD38 ＋ subpopulation that their augmented folds have significant difference compared with SFTI control group at the time culture around 6 weeks. The expanded CD34＋ cells could still form colonies CFU-GM, BFU-E and CFU-Mix over 12 weeks culture. Neither abnormality in karyotype analysis nor tumor was observed in nude mice from the expanded cells. Conclusions Our results demonstrate that human CB cells can be expanded for prolonged periods in culture by transduced JAK2 in response to AP20187 and cytokines combination. These findings have potentially useful application of cell therapy in treatment of genetically manipulated hematopoietic stern cell disorder.