Chen Yan, Lu Zhi-Gang, Han Ying
Department of Blood Transfusion, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2006 Jun;14(3):605-9.
This study was aimed to investigate the feasibility of cryopreserving red blood cells (RBCs) by loading with trehalose and to evaluate the effect of trehalose on lyophilized RBCs. Based on the thermal properties of RBC plasma membrane, the RBCs were incubated in 0.8 mol/L trehalose solution at 37 degrees C for 7 hours, and RBCs incubated in phosphate-buffered saline were used as control. The morphology of RBCs was observed by light and scanning electron microscopy, the hemolysis rate of loaded RBCs was detected by using cyanohemoglobin kit, the intracellular trehalose levels were assayed by sulfate anthrone method, the intracellular ATP and 2, 3-DPG levels were determined by bioluminescence assay and 2, 3-DPG kit respectively, meanwhile the deformation and osmotic fragility of RBCs were measured. The results showed that the intracellular trehalose concentration was 36.56 +/- 7.95 mmol/L, the electronical microscopic images of trehalose-loaded RBCs showed the membrane integrity, the hemolysis rate in trehalose-loaded RBCs was 15.663 +/- 3.848%, while hemolysis rate in controlled RBC was 5.03 +/- 1.85% (P < 0.05). Maximum index of deformation in trehalose-loaded RBC was 0.0289 +/- 0.00738, while maximum index of deformation in control group was 0.1200 +/- 0.0121 (P < 0.05), The level of ATP in trehalose-loaded RBC was 2.67 +/- 0.54 micromol/gHb, while the level of ATP in control group was 5.22 +/- 1.10 micromol/gHb (P > 0.05). Osmotic fragility data showed that trehalose exerted osmotic protection on RBC. During loading period the level of 2, 3-DPG in trehalose-loaded RBC was maintained close to the level in control. When trehalose-loaded RBCs were lyophilized and rehydrated, the recovery rate of hemoglobin was about 46.44 +/- 4.14% and that in control was 8.33 +/- 2.34% (P < 0.001). The recovery rate of hemoglobin of trehalose-loaded RBC was higher than that of control. It is concluded that trehalose can be integrated in the membrane of RBC in lyophilization state, maintain the integrity of RBC membrane, and significantly enhance the recovery rate of hemoglobin of cryopreserved RBCs. Cryopreserving RBCs by loading with trehalose is feasible.
本研究旨在探讨通过负载海藻糖冷冻保存红细胞（RBC）的可行性，并评估海藻糖对冻干红细胞的影响。基于红细胞质膜的热特性，将红细胞在37℃的0.8mol/L海藻糖溶液中孵育7小时，以在磷酸盐缓冲盐水中孵育的红细胞作为对照。通过光学显微镜和扫描电子显微镜观察红细胞的形态，使用氰化血红蛋白试剂盒检测负载红细胞的溶血率，采用硫酸蒽酮法测定细胞内海藻糖水平，分别通过生物发光测定法和2,3 - DPG试剂盒测定细胞内ATP和2,3 - DPG水平，同时测量红细胞的变形性和渗透脆性。结果显示，细胞内海藻糖浓度为36.56±7.95mmol/L，负载海藻糖的红细胞的电子显微镜图像显示膜完整性良好，负载海藻糖的红细胞的溶血率为15.663±3.848%，而对照红细胞的溶血率为5.03±1.85%（P<0.05）。负载海藻糖的红细胞的最大变形指数为0.0289±0.00738，而对照组的最大变形指数为0.1200±0.0121（P<0.05），负载海藻糖的红细胞中的ATP水平为2.67±0.54μmol/gHb，而对照组中的ATP水平为5.22±1.10μmol/gHb（P>0.05）。渗透脆性数据表明海藻糖对红细胞具有渗透保护作用。在负载期间，负载海藻糖的红细胞中2,3 - DPG水平维持在接近对照水平。当负载海藻糖的红细胞冻干并复水后，血红蛋白的回收率约为46.
