UM171+SR1结合细胞分群体外高效扩增造血干细胞的探究

Use of UM171/SR1 with cell clustering for highly efficiency expansion of hematopoietic stem cells in vitro

目的:探究UM171+SR1结合细胞分群体外高效扩增造血干细胞,获取可稳定体外长期扩增造血干细胞的方法。方法:将正常脐带血来源的造血干细胞分为4组,运用不同体系进行培养。对照组:StemSpanTMSFEM Ⅱ培养基+SCF+TPO+Flt3-ligand+IL-6,组1:StemSpanTMSFEM Ⅱ培养基+SCF+TPO+Flt3-ligand+IL-6+UM171,组2:StemSpanTMSFEM Ⅱ培养基+SCF+TPO+Flt3-ligand+IL-6+SR1,组3:StemSpanTM SFEM Ⅱ培养基+SCF+TPO+Flt3-ligand+IL-6+UM171+SR1。观察在不同细胞因子组合及结合后期流式细胞术分群培养对长期维持CD34+CD133+造血干细胞的增殖能力和干性等方面的影响。结果:在培养5天后,组3的CD34+CD133+造血干细胞数量(14.050±2.049)×106及CD34+CD133+百分比(66.13±4.603)%高于其他3组,CFU检测显示组3集落数目为(88.00±4.000),高于组1(64.67±3.215)及组2(51.33±6.506),均为P<0.05。后期细胞分群培养结果H+H组的细胞增值数目(38.44±0.4561)×106与CD34+CD133+阳性率(51.37±2.859)%明显高于其余实验组(P<0.05)。结论:造血干细胞的体外扩增培养过程中,在同时使用UM171和SR1的过程中结合细胞分群,且CD34+CD133high细胞群在细胞增殖及干性维持中发挥一定作用,可为造血干细胞的体外高效扩增培养提供可行的思路。

Objective:To explore highly efficient expansion of hematopoietic stem cells(HSC)in vitro using UM171+SR1 with cell clustering,so as to determine on a method for stable and long-term expansion of HSC in vitro.Methods:Normal umbilical cord blood-derived HSCs were divided into four groups cultured in different systems including:control group(StemSpanTMSFEM Ⅱ medium + SCF + TPO + Flt3-ligand + IL-6),group 1(StemSpanTMSFEM Ⅱ medium+SCF+TPO+Flt3-ligand+IL-6+UM171),group 2(StemSpanTMSFEM Ⅱ medium+SCF+TPO+Flt3-ligand+IL-6+SR1),and group 3(StemSpanTMSFEM Ⅱ medium+SCF+TPO+Flt3-ligand+IL-6+UM171+ SR1). The effects of different cytokine combinations and subsequent culture with flow cytometry clustering on long-term maintenance of the proliferative capacity and stemness of CD34+CD133+ HSCs.Results:After 5 days of culture,the number[(14.050± 2.049)× 106 ]and percentage[(66.13± 4.603)%]of CD34+CD133+ HSCs in group 3 were higher compared with the other three groups. CFU test showed that the number of colonies in group 3 was(88.00±4.000),which was higher than that in group 1(64.67±3.215)and group 2(51.33±6.506)(bothP< 0.05). In subsequent cell clustering,the number of proliferating HSCs[(38.44±0.456 1)×106]and the positive rate of CD34+CD133+ HSCs[(51.37±2.859)%]were significantly higher in the H+H group compared with the other experimental groups(P<0.05).Conclusion:During in vitro expansion and culture of HSCs,combined use of UM171 and SR1 plus cell clustering to focus on CD34 + CD133 highcell population plays a role in maintenance of HSC cell proliferation and stemness. This offers feasible clues to highly efficient expansion of HSCs in vitro.