海藻糖负载对人红细胞冷冻干燥保存影响的实验研究

Effects of trehalose loading on the freeze-dried red blood cells

目的初步探讨红细胞冻干长期保存的有效方法,并比较冻干前海藻糖的负载与否对红细胞冻干保存效果的影响。方法实验设实验组(负载海藻糖冻干-复水后红细胞):37℃,红细胞负载海藻糖7h后,采用主要成分为含有15%聚乙烯吡咯烷酮(PVP)和150mmol/L,海藻糖的缓冲液作为保护液,在设定的降温程序下冻干保存红细胞;对照组:未负载海藻糖冻干-复水后红细胞。冻干结束后用37℃的再水化液快速水化,检测2组的各项理化指标。结果实验组红细胞冻干再水化后RBC和Hb回收率要高于对照组(P<0.05);ATP酶和葡萄糖-6-磷酸脱氢镁(G-6-PD)活性水平显著差异有统计学意义(P<0.05))。结论胞内海藻糖对红细胞冻干具有明显的保护作用,红细胞在37℃孵育7h的条件负载海藻糖后冻干-复水后能保持细胞的理化稳定性。

Objective To find an efficient way for long-term preservation of red blood cells, and to evaluate the recovery and biological activities of the trehalose loaded and non-trehalose loaded RBCs after freeze-drying. Methods The samples were divided into two groups： the control group （without loading RBCs in trehalose buffer）, and the test group（loading RBCs in trehalose buffer）.We loaded RBCs at 37℃ for 7 hours, then put the RBCs into the protective solution, which was mainly composed of 150mmol/L trehalose, 15% polyvinylpyrrolidone （PVP） and isotonic buffer, and then lyophilized red blood cells by designed procedures. After freeze-drying, the samples were quickly rehydrated by 37℃ rehydration solution. RBCs recovery and the activity levels of ATP and G-6-PD were investigated. Results The recovery of RBCs with or without intracellular trehalose after freeze-drying and rehydration were over 70%, and there was no significant difference in MHC, MCHC between the two groups and the fresh RBCs. Morphological study also confirmed the results. The activity levels of ATP and G-6-PD of the test group were maintained close to those of fresh RBCs, which were significant different from the control group （P〈0.05）. Conclusion Trehalose has protective effect on freeze-drying RBCs, and the survival and biochemical stability of freeze-dried RBCs depend on both extracellular and intracellular vitrification.The study provide an important first step in long-term presservation of RBCs.