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0.25mol/L海藻糖组、5%二甲亚砜联合3%羟乙基淀粉及0.25mol/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。
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.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2011年第19期3602-3606,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
Joint Special Fund of Yunnan Provincial Science and Technology Committee & Kunming Medical College,No.2007C0036R~~
