联合海藻糖和二甲基亚砜进行冻干前负载的人红细胞冻干效果研究

Study of Freeze Drying of Human RBCs Loaded Trehalose and DMSO

探讨人红细胞在冻干前经添加低浓度二甲基亚砜(DMSO)海藻糖负载缓冲液负载后,是否会提高红细胞的冻干效果。实验组:为6%DMSO+800 mmol/L海藻糖。对照组:冻干前负载缓冲液为等渗PBS。取等量压积红细胞(6份)分别重悬于两组负载缓冲液,37℃孵育8 h后离心去除上清,加入冻干保护剂混匀后程序冻干,15%PVP溶液37℃三步复水。分别检测并比较两组冻干红细胞复水后血红蛋白回收率、复水红细胞变形性、再水化后4℃放置时间对冻干红细胞再水化稳定性的影响及冻干红细胞长期保存的稳定性。结果表明:冻干红细胞再水化后,实验组红细胞血红蛋白回收率为(63.019±4.901)%,对照组为(28.787±7.514)%,两组之间存在显著差异(P<0.01);冻干红细胞再水化后,两组冻干红细胞的综合变形指数均有明显下降,实验组红细胞综合变形指数为(18.133±2.392)%,对照组为(8.017±1.934)%,两组之间存在显著差异(P<0.01);两组冻干红细胞复水后4℃保存4 h、6 h、8h后,相对即刻再水化血红蛋白回收率的差值,经配对t检验显示有显著统计学差异(P<0.05)。复水后4℃保存12 h、24 h后,相对即刻再水化血红蛋白回收率的差值,经配对t检验显示无统计学差异;两组冻干红细胞4℃保存6个月前后复水后血红蛋白回收率的差值,经配对t检验显示两者之间存在显著差异(P<0.05)。结论:红细胞冻干前经添加6%DMSO的海藻糖负载缓冲液处理后,可提高冻干后红细胞血红蛋白回收率、改善复水红细胞变形性,提高复水后4℃保存的稳定性和冻干红细胞长期保存的稳定性。

To study whether can increase the effect of freeze drying when human red blood cells （RBCs） were incubated in trehalose loading buffer adding low concentration dimethylsulfoxide （DMSO） before lyophilization. The samples were divided into two groups ： the control group （loading buffer was PBS ） , and the test group （adding 6% DMSO to the 800 mmolfLtrehalose loading solution） . After incubated at 37℃ for 8 hours and freeze dried in program,we used 15% PVP solution rehydrated. Compared two group RBCs with hemoglobin recovery, RCD, the stability of rehydrated RBCs when stored in 4℃ and long-term preservation of lyophilized RBCs. The results showed that the rehydrated-lyophilized RBCs of tese group which hemoglobin recovery was （63. 019 ± 4.901 ）% and control group was （28. 787 ±7. 514）% （P 〈0.01 ）; that RCD of rehydrated-lyophilized RBCs decreased obviously which of test group was （ 18. 133 ±2. 392）% but control group was （8. 017 ± 1. 934）% （P 〈 0.01 ）. After rehydrated-lyophilized RBCs stored in 4℃, contrast to the baseline hemoglobin recovery of 4 h,6 h and 8 h had statistical difference between two groups （ P 〈 0.05 ） but which of 12 h and 24 h showed no difference. Contrast to the baseline hemoglobin recovery of lyophilized RBCs stored for 6 month in 4℃ had statistical difference between two groups （P 〈0.05）. It is concluded that after handled with trehalose-loading buffer adding 6% DMSO, rehydratedlyophilized human RBCs can increase hemoglobin recovery of RBCs, improve RCD, enhance the stability of rehydrated RBCs when stored in 4℃ refrigerate and long-term preservation of lyophilized RBCs.