SR-1/UM171在beta-地中海贫血来源的造血干细胞长期培养中对于细胞增殖和干性维持的作用研究

Effect of SR-1/UM171 on proliferation and stemness maintenance of hematopoietic stem cells from beta-thalassemia patients in long term culture

目的:探索一次性制备基因修饰自体造血干细胞产品的可能性.方法:将β-地中海贫血患者来源的造血干细胞分为五组, Con组: X-VIVO 20完全培养基;组1: SR-1 + X-VIVO 20完全培养基;组2: UM171 +X-VIVO 20完全培养基;组3: SR-1 +UM171 + X-VIVO 20完全培养基,组4为分选后冻存的CD34+细胞.观察不同的细胞因子或组合对长期培养中CD34+细胞增殖能力、干性维持等的变化.结果:在84小时培养时,组3的CD34+细胞百分比最高,达(89.8±4.8)%,细胞数目为(4.13±0.61)×10^6, CFU检测示其集落形成能力(177.7±5.5)与组4 (178.3±9.5)无统计学差异,小鼠植入试验表明组3和组4在3个月和6个月时,植入能力无统计学差异.超过84小时,无论采用何种方法培养CD34+细胞,其CD34+百分比及集落形成能力均逐渐下降.结论: SR-1、 UM171、 UM171+SR1等细胞因子或组合,均能在造血干细胞增殖的同时,维持干性. 84小时培养, UM171+SR1对于造血干细胞的干性维持效果最佳,也能较好地促进增殖,适合用于基因治疗的细胞培养.

Objective: To explore the possibility of one-step production of genetically modified autologous hematopoietic stem cells. Methods: The hematopoietic stem cells from beta thalassemia patients were divided into five groups: Con group (X-VIVO 20 complete medium), Group 1(SR-1 plus X-VIVO 20 complete medium), Group 2 (UM171 plus X-VIVO 20 complete medium), Group 3 (SR-1 plus UM171 and X-VIVO 20 complete medium), Group 4 (Cryopreserved CD34+cells after separation). The cell number, phenotype and colony formation ability were compared among different?groups. Results: In 84 hours, the percentage of CD34+cells in group 3 was the highest (89.8 ± 4.8)%, and the number of cells was (4.13±0.61)×10^6. There was no significant difference between group 3 and group 4 on colony formation ability (177.7±5.5 vs 178.3±9.5) and chimerism rate in NSI mice on third and sixth month. The phenotype and colony formation ability of each cultured group declined after 84 hours. Conclusion: Either cytokines or combination can maintain stemness of CD34^+cells from beta thalassemia patients and promote these cells proliferation. Group 3 (SR-1 plus UM171 and X-VIVO 20 complete medium) is the best option to maintain stemness of CD34^+cells and promote proliferation in 84 hours. This method can be applied for gene therapy.