Quan Guo Bo, Liu Min Xia, Ren Su Ping, Zhang Jin Gang, Han Ying
Institute of Transfusion Medicine, Beijing 100850, China.
Cryobiology. 2006 Aug;53(1):107-18. doi: 10.1016/j.cryobiol.2006.03.014. Epub 2006 Jun 9.
The plasma membrane of red blood cells permits sugars to be loaded into the cytoplasm simply by incubation in a suitable buffer solution containing the sugar. This may provide some hope for the freeze-drying of human red blood cells. However, the effect of the loading process on red blood cells has not been fully investigated. The exposure of phosphatidylserine (PS) on the surface of the cell can be recognized by macrophages and result in shortened circulation in vivo. This study evaluates the effects of the concentration, the incubation time, and the temperature of exposure of human red blood cells to extracellular trehalose or glucose. Exposure of PS was demonstrated by annexin V labeling. It was shown that the efficiency of loading of glucose was significantly greater than that of trehalose. The loading efficiency of both sugars increased with increase in extracellular sugar concentration, prolongation of incubation time, and increase of incubation temperature. The percentages of cells with exposed PS and of damaged cells were dependent on the extracellular sugar concentration, the incubation time, and the temperature. With an extracellular glucose concentration of 0.8M, the percentage of cells with exposed PS was more than 80% and significantly higher than that of red blood cells loaded with trehalose (approximate 20%, P<0.01). As the incubation time was prolonged, the percentage of PS exposure and of damaged cells also increased. After incubation for 5h, the percentage of red cells with exposed PS following loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (40%, P<0.01). In addition, the incubation temperature had a major effect on PS exposure. The percentage of cells with PS exposure and the proportion of damaged cells increased with increase of incubation temperature. At 37 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (P<0.01). However, when the temperature was below 25 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose or trehalose were both less than 10%. In conclusion, the loading efficiency for glucose was higher than that for trehalose, but the lesser effect of trehalose on exposure of PS suggests that it can maintain the asymmetrical distribution of membrane phospholipids and the intracellular trehalose can increase the osmotic tolerance of cells.
红细胞的质膜允许通过在含有该糖的合适缓冲溶液中孵育，将糖加载到细胞质中。这可能为人类红细胞的冻干提供一些希望。然而，加载过程对红细胞的影响尚未得到充分研究。细胞表面磷脂酰丝氨酸（PS）的暴露可被巨噬细胞识别，并导致其在体内循环时间缩短。本研究评估了人红细胞暴露于细胞外海藻糖或葡萄糖时的浓度、孵育时间和温度的影响。通过膜联蛋白V标记证明PS的暴露。结果表明，葡萄糖的加载效率显著高于海藻糖。两种糖的加载效率均随着细胞外糖浓度的增加、孵育时间的延长和孵育温度的升高而增加。PS暴露细胞和受损细胞的百分比取决于细胞外糖浓度、孵育时间和温度。当细胞外葡萄糖浓度为0.8M时，PS暴露细胞的百分比超过80%，显著高于加载海藻糖的红细胞（约20%，P<0.01）。随着孵育时间的延长，PS暴露和受损细胞的百分比也增加。孵育5小时后，加载葡萄糖后PS暴露的红细胞百分比超过80%，显著高于加载海藻糖的细胞（40%，P<0.01）。此外，孵育温度对PS暴露有主要影响。PS暴露细胞的百分比和受损细胞的比例随着孵育温度的升高而增加。在37℃时，加载葡萄糖后PS暴露细胞和受损细胞的百分比超过80%，显著高于加载海藻糖的细胞（P<0.01）。然而，当温度低于25℃时，加载葡萄糖或海藻糖后PS暴露细胞和受损细胞的百分比均小于10%。总之，葡萄糖的加载效率高于海藻糖，但海藻糖对PS暴露的影响较小，这表明它可以维持膜磷脂的不对称分布，并且细胞内的海藻糖可以增加细胞的渗透耐受性。
