造血干细胞低温保存与临床应用

目的观察低温保存造血于细胞（hematopoietic stem cell，HSC）的效率及冻存HSC的临床应用情况。方法25例骨髓造血干细胞（bone marrow stem cell，BMSC）以10％二甲基亚砜加10％自体血浆为冷冻保护剂，采用程控降温液氮保存。21例外周血于细胞（peripheral blood stem cell，PBSC）采用CP1（cryopreservativesl）加4％人血白蛋白为冷冻保护剂，非程控降温后液氮保存或低温冰箱保存。检测复温后单个核细胞（MNC）回收率、粒-单细胞集落形成单位（CFU—GM）回收率、台盼蓝拒染率（TBR），并行细菌学培养，移植后观察输注反应及植入情况。结果BMSC冻存时间为1～3个月，中位时间1个月。复温后MNC回收率为（97．74±0．85）％，TBR为（96．74±0．91）％，CFU—GM回收率为（72．04±1．87）％。PBSC冻存时间为1～20个月，中位时间2个月。复温后MNC回收率为（97．75±1．34）％，TBR为（96．28±2．16）％。复温后46份标本细菌学培养均为阴性。实施自体骨髓造血干细胞移植（ABMSCT）25例，中性粒细胞绝对值（ANC）〉0．5×10^9／L的时间为（10．79±0．93）d，血小板计数（PLT）〉20×10^9／L的时间为（12．88±0．95）d。实施自体外周血造血干细胞移植（APBSCT）21例，ANC〉0．5×10^9／L的时间为（10±1．14）d，PLT〉20×10^9／L的时间为（12．04±2．13）d。两组间MNC回收率和TBR比较均无显著性差异，移植后APBSCT组ANC〉0．5×10^9／L的时间早于ABMSCT组，有显著性差异（P〈0．05），但PLT〉20×10^9／L的时间两组无显著差异。出院前所有患者复查骨髓均示增生活跃或明显活跃。BMSC输注出现的不良反应明显多于PBSC输注。结论采用联合冷冻保护剂、非程控降温后液氮保存或低温冰箱保存来冻存HSC是切实可靠的，这种方法操作简便、价格低廉、安全有效；建议今后冻存BMSC前应去除红细胞。

Cryopreservation and clinical application of hematopoietic stem cells

Objective To investigate the effectiveness and clinical application of hematopoietic stem cell （HSC） cryopreservation. Methods 25 units of bone marrow stem cell （BMSC） were frozen by a programmed freezer and preserved in liquid nitrogen with 10% DMSO plus 10% autologous plasma as cryopreservative. 21 units of peripheral blood stem cell （PBSC） were frozen at an uncontrolled rate and preserved in liquid nitrogen or -80℃ refrigerator with CP1 plus 4% human album as cryopreservative. Mononuclear cell （MNC） recovery, granulocyte - monocyte colony forming unit （ CFU - GM） recovery,typeran blue resistance （TBR） and bacteriological culture were detected after thawing. Adverse reactions after infusion of cryopreserved HSC were observed and informations for engraftment were also collected. Results The median duration of BMSC cryopreservation was one month （1 - 3 months）. MNC recovery was （97.74 ± 0. 85） % （95.5% ± 99.5%）,TBRwas （96.74±0.91） % （94.5%±98%）,CFU- GM recovery was （72.04±1.87） % （69.5%±76. 5% ）. The median duration of PBSC cryopreservation was two months （1 -20 months）. MNC recovery was （97.75 ±1.34） % （96% - 99.5% ） ,TBR was （96.28 ± 2.16） % （93.5% - 99.5 % ）. 46 specimen were tested for germs by bacteriological culture,and all of them was negative. 25 units of cryopreserved BMSC were grafted to 25 patients （ autologous bone marrow stem cell transplantation,ABMSCT）. Among them the average time for absolute neutrophil count （ANC） 〉 0. 5 × 10^9/L was 10.79 ±0.93d （ 10 - 13d）and the average time for platelet count （PLT） 〉20 × 10^9/L was 12.88 ±0.95d （ 12 - 15d） after transplantation. 21 units of cryopreserved PBSC were used for autologous peripheral blood stem cell trans-plantations （APBSCT）. The average time for ANC 〉0.5 × 10^9/L was 10 ± 1.14d （8 - 12d） ,and the average time for PLT 〉 20 × 10^9/L was 12.04 ±2.13d （8 - 17d） after APBSCT. The differences of both MNC recovery and TBR be