糖基化修饰血小板冷藏稳定性研究

Stability of Glycosylated Platelets under Cold Storage

本研究旨在观察尿苷二磷酸半乳糖(UDP-Gal)糖基化修饰人血小板的稳定性、理化指标及体外功能变化。实验分为室温对照组、冷藏对照组以及修饰组(U+4组和4+U组)。机采人浓缩血小板悬液,4℃保存前或后添加适量UDP-Gal,进行糖基化修饰,分别于0、1、3、5、7、14天,通过荧光标记识别血小板膜糖蛋白末端半乳糖残基的凝集素(FITC-RCAⅠ)检测糖基化修饰效果;pH计检测血小板悬液pH值;血细胞计数仪检测血小板平均体积;比浊法测定血小板聚集率;流式细胞术检测血小板活化标志CD62P、血小板膜蛋白CD42b及PS的表达。结果表明:保存14天时,修饰组血小板RCAⅠ结合率是室温组的5-6倍;修饰组血小板悬液pH低于室温组,但二者之间无显著性差异(p>0.05);保存14天时室温组和修饰组血小板平均体积分别为10.6±1.9fL和11.14±1.1fL,二者之间无显著性差异(p>0.05);各组体外聚集活性在保存过程中均逐渐下降,但修饰组优于室温组(p<0.05);流式检测结果显示,保存1-5天修饰组CD62P、CD42b和PS表达的阳性率与新鲜血小板无显著性差异(p>0·05),但保存14天时,CD62P和PS表达的阳性率升高,CD42b表达的阳性率降低,与新鲜血小板相比差异极显著(p<0.01)。结论:在修饰血小板保存过程中,糖基化修饰的效果稳定,修饰血小板的pH和平均体积均处于正常值范围之内,聚集活性良好,优于室温保存组,但在保存5天后表现出不同程度的活化。

This study was aimed to investigate the stability and in vitro function of glycosylated platelets concentrates after long-term refrigeration. The experiments were divided into 4 groups： group preserved at room temperature （ RT group）, group preserved at 4℃ （4T group）, group glycosylated and preserved at 4℃ （U ＋ 4 group） and group preserved at 4℃ and glycosylated （4 ＋ U group）. All groups followed for up to 14 days. The binding rate of RCA I lectin and expression of Pit surface markers CD62P, CD42b and Annexin V binding were determined by flow cytometry. pH and mean volume were determined by pH meter and hematotocytometer respectively. Platelet aggregation was detected by aggregometer. The results showed that during storage up to 14 days RCA I binding rate of modified groups was 5 -6 fold of RT group. The pH of platelets suspension had no significant difference between these two groups（p 〉 0.05）. Mean volumes of both groups（ 10.6 ± 1.9 fL and 11. 14 ± 1. 1 fL） were also no significant difference（p 〉 0. 05）. Furthermore, aggregation responsiveness of modified groups was better than that of RT groups （p 〈 0.05 ） although both decreased during the storage. The expression level of CD62P, CD42b and Annexin V binding during 5 days of storage had no significant difference between modified and fresh platelet groups（p 〉0.05）. While the expression level of CD62P and PS increased and the expression level of CD42b decreased during storage up to 14 days, there was significant difference between modified and fresh platelet groups （p 〈 0.01 ）. It is concluded that the glycan modification is stable during storage up to 14 days. The glycosylated platelets retain in vitro function better than RT platelets during storage, but it shows activation to varying degrees in vitro after storage for 5 days.