人冻干血小板复水程序的优化

目的探讨不同复水条件对冻干血小板功能的影响。方法经过添加激活抑制剂(PGE1、左旋精氨酸、植酸钠和百维利肽)、DMSO和海藻糖等低温保护剂、冷冻干燥后获得冻干血小板,在一系列不同复水程序下对冻干血小板复水,应用流式仪检测并分析其CD62p和PAC-1的表达,评价血小板活化状态。通过正交设计,研究不同复水程序包括复水溶液、复水温度、复水方式3个影响因素对冻干血小板复水后活性的影响。结果 3个对冻干血小板复水后活化的影响因素排序:复水温度>复水方式>复水液(P<0.05);37℃条件下冻干血小板复水后血小板膜蛋白CD62p和PAC-1活化率最低,分别为(5.96±2.51)%和(4.55±1.97)%;预水化后再行四步添加复水可降低冻干血小板膜蛋白CD62p和PAC-1的表达率至(6.68±2.62)%和(5.45±2.06)%;添加聚乙烯吡咯烷酮(PVP)和血小板抑制剂的2号复水液对冻干血小板复水后膜蛋白CD62p和PAC-1的表达率影响最低,分别为(6.72±3.35)%和(5.28±2.30)%。总体模型评价进一步显示,第5实验组,即在37℃条件下加入PVP和血小板抑制剂的复水液配方,先预水化后再行四步法添加复水,其复水血小板活化率最低,CD62p和PAC-1的表达率分别为(3.06±1.36)%和(2.70±0.84)%。结论 37℃下,饱和蒸汽环境中预水化后,采用加入PVP及血小板抑制剂的复水液四步添加复水,其复水血小板活化率最低。

Preliminary study on the rehydrated conditions for lyophilized human platelets

Objective To explore the effect of different rehydration conditions on function of the lyophilized platelets.Methods After the lyophilization,platelets were given different rehydration conditions including different formula of rehydration solution,the rehydration temperature and the addictive method of rehydration solution using the orthogonal design.The expression of CD62p and PAC-1 were measured as the indexes of platelet activation.Results The impact of 3 factors from large to small order on viability of freeze-dried platelets was rehydration temperature,addictive method of rehydration solution and formular of rehydration solution,which all showed significantly different(P<0.05).If freeze-dried platelets were rehydrated under 37℃,the expression of membrane protein CD62p and PAC-1 was the lowest,which was(5.96±2.51) % and(4.55±1.97) %,respectively.If the freeze-dried platelets were rehydrated by the vapor first and then rehydration solution by 4 steps,the expression of platelet membrane protein CD62p and PAC-1 was also reduced,which was(6.68±2.62) % and(5.45±2.06) %,respectively.If freeze-dried platelets were rehydrated with the second rehydration solution with polyvinylpyrrolidone(PVP) and platelet inhibitors,the expression of membrane protein CD62p and PAC-1 was the lowest,which was(6.72±3.35) % and(5.28±2.30) %,respectively.The overall evaluation model also showed that the 5th experimental groups of freeze-dried platelets was optimal whose procedure was the rehydration solution with PVP,and platelet inhibitors and rehydrated by the vapor first,and then rehydration solution by 4 steps under 37℃.After rehydration,the activation of platelets was the lowest,with the expression of CD62p and PAC-1 of(3.06±1.36)% and(2.70±0.84)%,respectively.Conclusion The experimental results showed that with preliminary hydration in 37℃ saturated steam environment,using rehydration solution combined with PVP and platelet inhibitors 4 steps adding procedure,rehydrated platelets showed the lowest platelet activation rate.But whether as freeze-dried platelets rehydration program recommended,we still need further experimental studies.