海藻糖负载红细胞及其冻干保存研究

为了研究海藻糖负载红细胞方法的可行性及红细胞内海藻糖对冻干红细胞的影响,利用红细胞膜在37℃时细胞膜上部分脂质由固态变为液态、流动性增大和膜通透性增加的性质,将红细胞置于高浓度海藻糖负载液中孵育7小时,并以磷酸缓冲盐溶液中孵育的红细胞作为对照,对红细胞的海藻糖负载率、形态学、渗透脆性、变形性、ATP含量及2,3-DPG含量进行评价。结果表明:负载后红细胞内海藻糖含量为36.56±7.95mmol/L,实验组红细胞溶血率为(15.663±3.848)%,对照组红细胞溶血率为(5.03±1.85)%,差异显著(P<0.05);实验组红细胞变形指数是0.0289±0.00738,对照组红细胞变形指数是0.1200±0.0121,差异显著(P<0.05);负载后实验组红细胞内ATP含量为2.67±0.54μmol/gHb,对照组红细胞内ATP含量为5.22±1.10μmol/gHb(P>0.05),实验组红细胞渗透脆性降低,明显低于对照组。尽管负载组的红细胞大小不一,形态各异,但在透射电镜下绝大多数红细胞膜完整,胞内血红蛋白密度均匀,而对照组有近一半的细胞膜不完整并有漏孔,胞内血红蛋白密度变浅。实验组与对照组中2,3-DPG含量均为零。实验组红细胞冻干再水化后,血红蛋白回收率46.44±4.14%,对照组血红蛋白回收率8.33±2.34%,差异显著(P<0.001)。结论:海藻糖负载的红细胞功能符合输注标准,负载方法可行,负载入细胞内的海藻糖能够保持细胞膜的完整性,大大提高了冻干红细胞的回收率,为红细胞的冷冻干燥成功迈出了第一步。

Trehalose Loading Red Blood Cells and Freeze-drying Preservation

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.