深低温冻存的外周血单个核细胞活性氧水平与造血干/祖细胞归巢黏附分子表达之间关系的研究

本研究通过检测经程控降温液氮保存前后的富集造血干/祖细胞的外周血单个核细胞(PBMNC)的活性氧(ROS)水平及造血干/祖细胞归巢分子的表达,分析二者之间的相关性。以冻存PBMNC为实验组,以新分离PBMNC为对照组,通过台盼蓝染色方法检测2组细胞的存活率;通过流式细胞分析法检测2组细胞ROS水平;通过流式细胞分析法检测造血干/祖细胞表面抗原CD34、CD133以及归巢相关分子VLA4、CXCR4、CD44的表达;通过相关系数分析ROS水平与归巢分子表达之间的相关性。结果表明:PBMNC随冻存时间延长存活率逐渐降低,冻存3、6、9、12个月的细胞存活率比冻前显著降低(P<0.05);造血干/祖细胞表面标志性抗原CD34、CD133及归巢相关分子CD44、VLA4、CXCR4表达率随冻存时间的延长有所下降,冷冻保存1年后的表达率比冻存前显著降低(P<0.05);随着冷冻保存时间的延长,细胞内ROS水平逐渐升高,冻存6、9、12个月后细胞内ROS水平比冻存前显著升高(P<0.05)。PBMNC内ROS水平与造血干/祖细胞CD34+VLA4+、CD34+CXCR4+、CD34+CD44+双阳性表达率均呈负相关(相关系数分别为-0.503、-0.457、-0.465)。结论:造血干/祖细胞的归巢黏附分子表达随冻存时间延长而逐渐下降;冻存的PBMNC中的ROS水平与造血干/祖细胞归巢黏附分子表达率成显著负相关;ROS水平增高可能是冻存外周血造血干/祖细胞归巢功能降低的原因之一。

Correlation between Reactive Oxygen Species of Cryopreserved Peripheral Blood Mononuclear Cells and Expression of Homing Adhesion Molecules on Peripheral Blood Hematopoietic Stem/progenitor Cells

This study was purposed to detect the level of reactive oxygen species （ROS） in cryopreserved peripheral blood mononuclear cells （PBMNC） and the expression of homing molecules on peripheral blood hematopoietic stem/ progenitor cells, and to analyze the correlation between ROS level and the expression of homing molecules. The survival percentage of PBMNC was determined by trypan blue exclusion rate. The expression of antigens on peripheral blood hematopoietic stem/progenitor cells, such as CD34, CD133 and some related homing molecules （ VLA4, CXCR4 and CD44） were detected by flow cytometry. The correlation of the ROS levels with the expression of homing molecules was evaluated by the correlation coefficient. The results showed that the survival rate of PBMNC was gradually reduced along with prolongation of cryopreserved time, and there was a significant difference in the survival percentage of cryopreserved PBMNC between experimental group frozen for 3, 6, 9, 12 months and control group （ P 〈 0.05 ）. The expression of marked antigen CD34 and CD133 , and homing adhesive molecules of CD44, VLA4 and CXCR4 declined with the extension of frozen time, and there was significant statistical difference （ P 〈 0.05 ） between experimental group cryopreserved for a year and control group. The level of ROS in PBMNC gradually increased along with the prolongation of cryopreserved time, and there was significant statistical difference （ P 〈 0.05 ） between experimental group freezing- stored for 6, 9 and 12 months and control group. The correlation coefficients of ROS level and expression percentageCD34 ^＋ VLA4 ^＋ , CD34 ^＋ CXCR4 ＊, CD34 ^＋ CD44 ^＋ PBMNC were - 0. 50, - 0. 457, - 0. 465, respectively. It is concluded that ROS level in PBMNC increases significantly with the prolonged cryopreservation time, especially for more than 6 months. There is a significant negative correlation between the ROS level of PBMNC and the expression rate of homing molecules. Higher level of ROS may be one of the reasons for the reduction of homing capability in peripheral blood stem/progenitor ceils after cryopreservation.