深低温冷冻对脐带血干祖细胞影响的研究  

Effects of cryopreservation on cord blood stem progenitor cells

作　　者：贺雪萍 鲁瑞周 李茜 韩俊领 HE Xue-ping;LU Rui-zhou;LI Xi;HAN Jun-ling(Xiehe Stem Cell Genetic Engineering Co,Ltd,Tianjin 300384,China;Guizhou Heze Biotechnology Co,Ltd,Guiyang 550005,Guizhou,China)

机构地区：[1]协和干细胞基因工程有限公司,天津300384 [2]贵州和泽生物科技有限公司,贵州贵阳550005

出　　处：《生物医学工程与临床》2020年第3期252-256,共5页Biomedical Engineering and Clinical Medicine

摘　　要：目的探讨深低温冷冻对脐带血中干祖细胞产生的影响。方法取30份脐带血标本,经过羟乙基淀粉离心沉淀,去除成熟红细胞及血浆,收集有核细胞悬液,然后采用标准的程控降温冷冻方法:以(1~2)℃/min的降温速率进行程控降温,降到-80℃后放入-196℃液氮内保存,对有核细胞悬液进行冷冻保存。对冷冻前后的细胞悬液进行有核细胞总数(TNC)、CD34^+细胞、CD41^+细胞回收率检测,分析细胞活率及各系祖细胞集落形成单位表达情况。结果经过对脐带血标本一系列处理及深低温冷冻、融化后,脐带血中TNC回收率为92.57%,CD34^+细胞回收率为86.53%。冷冻前后各系祖细胞集落形成单位比较,巨核系祖细胞集落形成单位(CFU-Meg)的回收率(46.50%)要明显低于粒单系祖细胞集落形成单位(CFU-GM)的回收率(76.58%)(t=18.87,P <0.001)。对脐带血免疫表型检测,冷冻前脐带血有核细胞中CD34^+、CD41^+分别为(0.31±0.11)%、(0.05±0.01)%,冷冻后有所下降[(0.28±0.10)%、(0.03±0.01)%];CD34^+、CD41^+冷冻后的回收率分别为86.88%、62.12%;差异有统计学意义(t=7.01,P <0.05)。结论在目前的分离冷冻条件下,深低温冷冻对脐带血中各系祖细胞影响不同,巨核系祖细胞与粒单核系祖细胞相比,表现出对深低温冷冻更加敏感。Objective To investigate the effect of cryopreservation on stem progenitor cells in cord blood. Methods A total of30 umbilical cord blood samples were centrifuged with hydroxyethyl starch to remove mature red blood cells and plasma, and then nuclear cell suspension was collected. The nuclear cell suspension was cryopreserved by standard programmed cooling method, which programmable cooling temperature was-80 ℃ at temperature cooling rate of(1-2) ℃/min, and then stored in-196 ℃ liquid nitrogen. The total nucleated cell(TNC), CD34^+, CD41^+cells, cell viability and colony forming units of progenitor cells were detected before and after freezing. Results After specimens were processed and cryopreserved and thawed, the recovery rate of TNC in umbilical cord blood was 92.57 %, and recovery rate of CD34+was 86.53 %. Compared each of progenitor cell colony forming unit(CFU) before and after cooling, the recovery rate of colony forming unit megakaryocyte(CFU-Meg)(46.50 %) was significantly lower than that of colony forming unit-granulocyte and macrophage(CFU-GM)(76.58 %)(t = 18.87, P < 0.001). For umbilical cord blood immunophenotyping, CD34+and CD41+in umbilical cord blood nucleated cell were(0.31 ± 0.11) % and(0.05 ± 0.01) % before cooling, and decreased after cooling[(0.28 ± 0.10) %,(0.03 ± 0.01) %]. The recovery rate of CD34+and CD41+after cooling were 86.88 % and 62.12 %, respectively, and the difference was statistically significant(t = 7.01, P < 0.05). Conclusion It is demonstrated that in current isolation and freezing conditions, cryopreserve shows different effects on umbilical cord blood progenitor cells. The megakaryotic progenitor cells are more sensitive to cryopreservation, compared with granulocyte mononuclear progenitor cells.

关 键 词：脐带血 巨核系祖细胞 粒单核系祖细胞 深低温冷冻 

分 类 号：R457.7[医药卫生—治疗学]