利用SCID小鼠模型评价-80℃冷冻人血小板的体内生存能力

Evalution of In Vivo Viability of Human Platelets Cryopreserved at -80℃ by Using SCID Mouse Model

为了评估人血小板-80℃冷冻保存后在活体内的生存能力,取新鲜机采血小板加入二甲基亚砜(DMSO),使其终浓度为5%,于-80℃保存10天后,取出立即放置于37℃水浴箱中快速解冻,并离心浓缩10倍。用带有超细针头的胰岛素注射器抽取浓缩血小板100μl,经尾静脉注入重症联合免疫缺陷(SCID)小鼠体内,在注入0.5、2、4、6、12、24小时时采集小鼠全血,肝素抗凝,用CD61-PE标记后,流式细胞术计数人血小板,以30分钟时的人血小板计数为100%,计算人血小板存活率。结果表明:冷冻血小板在活体内存活率明显降低,新鲜血小板和冷冻血小板输注SCID小鼠体内4小时时的存活率分别为(79.5±9.1)%和(40.6±6.6)%(p<0.01),推算半寿期(T1/2)分别为7小时和2.5小时。结论:血小板-80℃冷冻保存后在活体内的生存能力降低。

The purpose of this study was to evaluate the in vivo viability of human platelets cryopreserved at - 80℃ by using SCID mouse model and flow cytometry. The fresh human platelets were frozen with 5% DMSO at - 80℃ for 10 days ,thawed, and centrifuged for concentration. A 100 ml aliquot of concentrated platelets was injected into the SCID mouse tail vein by using a 1 ml insulin-syringe fitted with a 29-gauge ultra-fine needle. The whole blood was collected into heparinized capillary tube at 0.5, 2, 4, 6, 12, and 24 hours after infusion via a tail vein and was labelled with CD61-PE. Then the human platelets in mouse whole blood were detected by flow cytometry. The 30 minute time point was used as 100% to calculate the survival time of human platelets. The results showed that the survival time of cryopreserved human platelets were more significantly decreased than that of fresh platelets in SCID mice. Survival rates at 4 hours after transfusion of fresh platelets and cryopreserved platelets in SCID mice were 79.5 % ±9. 1% （ n = 8） and 40. 6%± 6. 6% （n = 8 ） respectively, and a T1/2 estimated were 7 hours for fresh platelets, but 2. 5 hours for the cryopreserved. In conclusion, platelets survival time in SCID mice was shortened after frozen with DMSO at -80℃.