冻干保存血小板可逆性抑制激活的实验研究

A study on the reversible activation inhibitors for human platelets lyophlization

目的探索最优的血小板激活抑制剂组成方案,以建立一套完善的血小板冻干前处理程序。方法选择已基本确定有效作用浓度的6种血小板激活抑制剂如前列腺素E1、腺苷、左旋精氨酸、植酸钠、百维利肽和西洛他唑,结合血小板冷冻干燥保护剂的负载过程,以CD62p、PAC-1、MPV和P lt作为血小板活化指标,CD62p和PAC-1的再表达率作为血小板反应性指标,诱导剂诱导的血小板最大聚集率和SPAT作为血小板聚集和促凝血功能的指标;采用正交实验设计,分8个实验组,研究分析各因素在负载中对血小板状态和功能的影响,选择最优的负载液组成。结果A因素(负载环境)水平Ⅰ在抑制血小板MPV增大、D62p和PAC-1的表达以及再表达率保留、血小板最大聚集率和SPAT影响均优于水平Ⅱ(P均<0.05);B因素(PGE1)C因素(左旋精氨酸)F因素(植酸钠)G因素(百维利肽)的水平Ⅱ则均由于各自的水平Ⅰ。因此,在8个实验条件中,以第3实验组(A1B2C2D1E1F2G2)对血小板功能保护的最好。结论在血小板冻干前保护剂负载过程程序,在血浆负载环境中添加1μmol/L前列腺素E1,5 mmol/L左旋精氨酸,0.5 mmol/L植酸钠和0.5μmol/L百维利肽,可有效抑制血小板激活,使保存的血小板具有表达CD62p和PAC-1活性、聚集反应和促凝血活性。

Objective To seek the best composition of platelet activation inhibitors for lyophilization protection loading solution. Methods The expressions of CD62p PAC-1, MPV and Pit count were used as indexes of platelets activation. The reexpressions of CD62p and Pac-1 induced by thrombin were used as indexes for platelet reactivity. The maximal agg＇regation and SPAT were indexes for platelet aggregation function. Orthogonal experimental design was used for analysing six inhibitors and lyophilizing-protectant loading solution. Then the best combination of loading protectants was selected. Results The level 1 of factor A had no adverse effect on platelet MPV, CD62p, PAC-1, PAgT and SPAT. The level 2 of factor B, C, F and G could inhibit platelet activation and retain their functions. So the solution compound of the third group （Az B2C2D, El F2G2） was the best one. Conclusion Adding loading solution including plasma, 1umol/L prostaglandin E1,5mmol/L L-arginine, 0. 5mmol/L phytic acid and 0. 5 umol/L bivalirudin could prevent platelets from activation damages before lyophilization.