冻干前人红细胞对海藻糖的负载影响因素研究

Factors affacting trehalose loading in red blood cells before freeze-drying

目的研究影响海藻糖负载多种因素,探讨人红细胞对负载海藻糖的影响因素及规律性。方法根据红细胞海藻糖的负载量衡量,利用硫酸-蒽酮法检测红细胞在不同胞外海藻糖浓度、不同孵育时间、不同孵育温度的条件下对海藻糖的摄取量,并检测红细胞溶血程度。结果红细胞内海藻糖在负载液中的浓度<1000 mmol/L、负载时间<9 h、负载温度<37℃条件下,海藻糖的摄取量呈正相关。在负载液中浓度为0、200、400、600、800、1000mmol/L时,红细胞内海藻糖浓度分别为0、10.03、14.5、41.7、55.3和71.6 mmol/L;在温度为37℃时红细胞在浓度为1 000 mmol/L负载液中分别孵育0、1、3、5、7、9 h,胞内海藻糖浓度分别为0、5.73、6.11、55.7、和61.2 mmol/L。对温度、时间和胞外海藻糖浓度的统计分析显示,温度对负载后胞内海藻糖浓度的影响最大(P<0.01)。结论37℃、采用新鲜红细胞在海藻糖浓度为800 mmol/L的负载缓冲液中孵育7 h能有效摄取海藻糖,使之达到对红细胞起到冻干保护作用的胞内海藻糖理论浓度。

Object To study the factors that influence trehalose loading, and to discuss the due for loading RBCs with trehalose across to the intracellular trehalose. Methods RBCs were mixed with trehalose and incubated at different temperatures, incubation durations and different trehalose concentrations, and anthrone reaction was used to determine the amount of the intracellular trehalose, meanwhile we evaluated hemolysis of loaded RBCs. Results With extraceUular trehalose concentration of 〈 1 000 mmol/L, incubation duration 〈9 h and the temperature 〈37 ℃, trehalose loading in RBCs was correlated with the temperature, duration and extracellular trehalose concentration. When extracellular trehalose concentration were 0,200,400,600,800, and 1 000 mmol/L, trehalose loading in RBCs were 0,10.03,14.5,41.7,55.3 and 71.6 mmol/L, respectively ; When the incubation duration were 0,1,3,5,7,9 h, trehalose loading in RBCs were 0,5.73, 6.11,55.7and 61.2 mmol/L, respectively. For these factors, the statistical results showed that the temperature had the most effects on trehalose loading. Conclusion Fresh RBCs incubated with 800 mmol/L trhalose at 37℃ for a time span of 7 h results in the highest intracellular trehalose concentration of about 52 mmol/L. This study provides an important first step in the long-term preservation of RBCs.