中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (19): 3602-3606.doi: 10.3969/j.issn.1673-8225.2011.19.044

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

不同冷冻保护剂对低温冰箱转液氮阶梯降温冷冻保存造血干细胞效果的影响

杨同华，赵仁彬，蒋雅先，欧阳红梅，张爱玲，胡  芃，刘建琼，闻  艳   

  1. 昆明医学院附属昆华医院，云南省第一人民医院血液科，云南省昆明市  650032
  • 收稿日期:2011-01-10 修回日期:2011-03-31 出版日期:2011-05-07 发布日期:2011-05-07
  • 通讯作者: 赵仁彬，主管检验师，昆明医学院附属昆华医院，云南省第一人民医院血液科，云南省昆明市 650032
  • 作者简介:杨同华★，1964年生，云南省水富县人，汉族，1996年成都中医药大学毕业，硕士，主任医师，教授，硕士生导师，主要从事血液病诊治、造血干细胞临床移植和实验室研究工作。
  • 基金资助:

    云南省科技厅-昆明医学院联合专项资金资助项目(2007C0036R)。

Influences of different cryoprotectants by ladder-style freezing from low temperature refrigerator to liquid nitrogen on cryopreservation of hemopoietic stem cells

Yang Tong-hua, Zhao Ren-bin, Jiang Ya-xian, Ouyang Hong-mei, Zhang Ai-ling, Hu Peng, Liu Jian-qiong, Wen Yan   

  1. Department of Hematology, Kunhua Hospital of Kunming Medical College, the First People’s Hospital of Yunnan Province, Kunming  650032, Yunnan Province, China
  • Received:2011-01-10 Revised:2011-03-31 Online:2011-05-07 Published:2011-05-07
  • Contact: Zhao Ren-bin, Laboratorian-in-charge, Department of Hematology, Kunhua Hospital of Kunming Medical College, The First People’s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China zhaorenbin@126.com
  • About author:Yang Tong-hua★, Master, Chief physician, Professor, Master’s supervisor, Department of Hematology, Kunhua Hospital of Kunming Medical College, the First People’s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China ynanblood@yahoo.com.cn
  • Supported by:

    Joint Special Fund of Yunnan Provincial Science and Technology Committee & Kunming Medical College, No.2007C0036R*

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被引次数

1

摘要：

背景：非程序降温-80 ℃低温冰箱保存方便快捷，程序降温-196 ℃液氮保存可靠长久，将两者合二为一简化流程已成功用于临床。
目的：观察不同冷冻保护剂对-80 ℃低温冰箱转液氮阶梯降温冷冻保存造血干细胞效果的影响。
方法：分设10%二甲亚砜组、5%二甲亚砜联合3%羟乙基淀粉组、5%二甲亚砜联合0.25 mol/L海藻糖组、5%二甲亚砜联合3%羟乙基淀粉及0.25 mol/L海藻糖组。采用  -80 ℃低温冰箱转液氮阶梯降温法对单采外周造血干细胞进行冷冻保存，通过透射电镜观察细胞超微结构变化，流式细胞仪观察Annexin-V、PI、Caspase-3水平。
结果与结论：4组冷冻保存细胞的存活率、凋亡率和死亡率差异均无显著性意义(P > 0.05)。透射电镜下各组细胞超微结构变化差异不明显。单个核细胞群落冷冻保存后存活率在90%以上，含成熟细胞较多的CD45+细胞群落凋亡发生率可达50%左右。造血干祖细胞群落中，早期细胞较晚期细胞更能耐受冷冻损伤。提示在基础冷冻保护剂二甲亚砜的基础上，加入羟乙基淀粉和海藻糖并未显示出对冷冻保存效果的增强作用。

关键词: 冷冻保护剂, 凋亡, 造血干细胞, 程序降温, 非程序降温

Abstract:

BACKGROUND: Uncontrolled-rate freezing in -80 ℃ refrigerator is convenient, while controlled-rate freezing in -196 ℃ liquid nitrogen is reliable and long-term, the combination of the two can simplify the process and has been successfully used in clinics.
OBJECTIVE: To explore the influences of different cryoprotectants by ladder-style freezing from -80 ℃ low temperature refrigerator to liquid nitrogen on the cryopreservation of hemopoietic stem cells.
METHODS: The experiments were divided into four groups: 10% dimethyl sulfoxide (DMSO) group, 5% DMSO combined with 3% hydroxyethyl starch group, 5% DMSO combined with 0.25 mol/L trehalose group, 5% DMSO combined with 3% hydroxyethyl starch and 0.25 mol/L trehalose group. Peripheral hemopoietic stem cells were cryopreserved by ladder-style freezing from -80 ℃ low temperature refrigerator to liquid nitrogen. The ultrastructural changes were examined by transmission electron microscopy, the expressions of Annexin-V, PI and Caspase-3 were detected by flow cytometry.
RESULTS AND CONCLUSION: There was no significant difference in survival rate, apoptotic rate and necrotic rates of the cryopreserved cells in the four groups (P > 0.05). The ultrastructural changes had no significant difference under the transmission electron microscopy. The viability was more than 90% in frozen-thawed mononuclear cell colonies, and the apoptosis was roughly 50% in the frozen-thawed CD45+ cell population, which contained many mature cells. Of hemopoietic stem cells, early stage cells have greater resistance to damage of cryopreservation than late stage cells. It is concluded that the addition of hydroxyethyl starch or trehalose into DMSO exhibits no synergistic protective effect on the cryopreservation of hemopoietic stem cells.

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