脐血造血干细胞不同温度长期冻存效果的实验研究  

Study on long cryopreservation effect of cord blood hematopoietic stem cells under different temperature

作　　者：许利民[1] 王劲[1] 洪淋[1] 余昌云[1] 张二铁[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所血液科,重庆400042

出　　处：《中国现代医学杂志》2007年第24期3034-3036,3039,共4页China Journal of Modern Medicine

摘　　要：目的通过观察在不同温度条件下长期冻存的效果,以探讨脐血造血干细胞适宜的保存时间。方法以终浓度为5%二甲基亚砜(DMSO)、3%羟乙基淀粉(HES)和4%人血白蛋白(HSA)作为细胞冷冻保护剂,分别采用-80℃冰箱直接冻存和程控降温-196℃液氮冻存,冻存前及冻存3、6及12个月后,应用全自动血细胞分析仪测定脐血单个核细胞(MNC)计数,台盼蓝拒染法显微镜下计数细胞存活率及流式细胞仪测定CD34+细胞计数。结果在-80℃冰箱冻存3个月后,脐血MNC计数、台盼蓝拒染率及CD34+细胞计数冻存前后比较差异均无显著性。冻存6个月后,脐血台盼蓝拒染率较冻存前差异有显著性(P<0.05)。冻存12个月后,脐血各项指标较冻存前差异均有显著性,其中台盼蓝拒染率差异存在非常显著性(P<0.01)。在-196℃液氮冻存3、6及12个月后,脐血MNC计数、台盼蓝拒染率及CD34+细胞计数冻存前后比较均无显著差异。结论以终浓度为5%二甲基亚砜(DMSO)、3%羟乙基淀粉(HES)和4%人血白蛋白(HSA)作为细胞冷冻保护剂,脐血造血干细胞在-80℃冰箱中可以有效保存3个月,6个月后存在部分细胞损害,此时的脐血不适合作干细胞移植。脐血经程控降温-196℃液氮冻存12个月后,造血干细胞仍保存完好,适宜长期保存。[Objective ] To probe appropriate time of conservation of cord blood hematopoietic stem cells, by studying the effect of long cryopreservation under different temperature. [Methods] Cord blood hematopoietic stem cells were cryopreserved with a cryopmtectant agent composed of final density 5% dimethlsttlfoxide（DMSO）, 3% hydroxyethyl starch（HES） and 4% human serum albumin（HSA）, using -80℃ direct freezing procedure and -196℃ program-controlled freezing procedure separately. The number of cord blood mononuclear cells（MNC） were measured by automatic blood cells analyzer, the rate of cell livability was measured by trypan blue exclusion by microscope and the number of CD34^＋ cells was measured by flow cytometry, freezing before and after 3, 6 and 12 months. [Results] There was no difference on the number of MNC and CD34^＋ cells and the rate of cell livability when cord blood stem cells were cryopreserved in -80℃ refregerater for 3 months. There was significant difference on the rate of cell livability of cord blood stem cells, freezing for 6 months, compared with before freezing（P 〈0.05）. Each index of cord blood stem cells obviously changed pre-and-post freezing in -80℃ refregerater for 12 months, and there was very signitlcant difference on the rate of cell livability especially（P 〈0.01）. Cord blood hematopoietic stem cells were conserved in good condition freezing in -196℃ for 12 months. [Conclusion] It was suggested that, using 5% DMSO, 3% HES and 4% HSA as cryoprotectant agent, freezing in -80℃ refregerater, cord blood hematopoietic stem cells maybe conserve in good condition within 3 months, and there are some damages of cell livability of cord blood stem cells freezing for 6 months, at that time it is not suitable for cord blood transplantation. Cord blood hematopoietie stem cells were conserved still in good condition by program-controlled freezing under -196℃ for 12 months, it is suitable for long cryopreservation.

关 键 词：脐血 造血干细胞 冷冻保存 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]