人血小板冻干前负载海藻糖技术研究

A Study on Trehalose Loading Technique of Human Platelets before Freezing-dried

目的 建立血小板负载海藻糖基本技术 ,包括负载温度 ,负载时间和海藻糖负载浓度。方法 通过比较生理温度(3 7℃ )和相变温度 (16℃ )下血小板胞内海藻糖浓度与负载时间曲线 ,血小板平均体积 (MPV)与负载时间、海藻糖负载浓度曲线 ,优选负载率较高的负载温度和负载时间。在此基础上 ,做海藻糖负载浓度与血小板胞内浓度关系曲线 ,并计算海藻糖负载率 ,确定血小板负载海藻糖基本技术。结果 3 7℃海藻糖负载率明显高于 16℃负载 ,3 7℃负载 4h海藻糖负载率高达 11 6% ,16℃负载 4h海藻糖负载率为 5 6% ;血小板MPV在 16℃比 3 7℃明显增大 43 2 % ,但随负载时间和负载浓度改变不发生明显改变 ;3 7℃条件下负载 ,血小板MPV变化与负载时间和海藻糖负载浓度呈现正相关性 ,且负载时间与海藻糖负载浓度存在协同效应 ,海藻糖负载浓度低于 5 0mM ,负载时间在 1h～ 4h内 ,MPV没有明显变化 ,高于 5 0mM ,随浓度、负载时间增加而增大 ;3 7℃ ,4h负载海藻糖 ,海藻糖负载浓度为 45mM时 ,负载率最高达 13 86%。结论 负载温度 3 7℃ ,负载时间 4h ,负载浓度 45mM可以作为血小板海藻糖负载基本技术参数 ,负载浓度可根据冻干保存需要进行调整。

Objective To establish the technique of loading trehalose into human platelets including temperature, time and concentration of trehalose loading. Methods Intracellular trehalose concentration versus loading time curves at 37℃ and 16℃, mean plateletee volume(MPV) versus loading time curves, and loading concentration curves were investigated and compared. Then loading time and temperature for high loading efficiency of trehalose into human platelets were ascertained. Intracellular trehalose versus trehalose loading concentration curves were made, and loading efficiency was caculated. Results The trehalose loading efficiency of 4 hours at 37℃ was significantly higher than that at 16℃ (11.6% vs 5.6%). MPV at 16℃ increased 43.2% compared with at 37℃, but had no changes along with the change of loading time and concentration. When loading at 37℃, MPV change was positively related with loading time and concentration, and loading time and loading concentration had a cooperative effect. MPV had no changes in 1～4hours loading when trehalose loading concentration was under 50mM. MPV increased with the increase of loading time and concentration when trehalose loading concentration was above 50mM. When trehalose loading concentration was 45mM, loading efficiency at 37℃ reached 13.86% 4 hours aftr loading. Conclusion The basic parameter of trehalose loading technique of human platelets was loading temperature at 37℃, loading time for 4 hours, and loading concentration of 45mM.