人血小板冻干前负载海藻糖技术的研究

Process of Human Platelets Loaded with Rehalose before Lyophili-zation

为研究人血小板冻干前负载海藻糖技术与方法,筛选出最佳负载海藻糖实验条件并进一步研究血小板在温度37℃条件下、负载海藻糖4小时后的平均体积、体外激活程度和聚集反应性的变化,绘制血小板胞内海藻糖负载效率及浓度随温度、时间、胞外海藻糖浓度变化曲线,筛选合适的负载条件,分别以凝血酶、ADP、胶原、瑞斯托霉素4种物质作为血小板激活诱导剂,用血小板聚集仪分别检测血小板负载海藻糖前后的聚集反应性,用流式细胞仪检测分析血小板负载海藻糖前后其膜表面糖蛋白分子CD62p、PAC-1的表达率,加可逆性激活抑制剂PGE-1、腺苷后血小板激活被抑制的程度。结果表明:海藻糖负载效率与孵育时间(2小时后)、温度(30-40℃)呈良好线性关系,在37℃条件下经4小时孵育后负载效率可达60%,载入到胞内海藻糖浓度随胞外海藻糖浓度(<50mmol/L)的升高而递增;同负载前相比较,血小板平均体积(MPV)、血小板对4种激活诱导剂的最大聚集率均无显著性差别(P>0.01),经4小时负载海藻糖后血小板膜表面CD62p表达率升高,但联合添加可逆性激活抑制剂PGE-1、腺苷后CD62p表达率显著下降。结论:37℃、4小时、胞外海藻糖浓度小于50mmol/L为合适负载条件,添加可逆性血小板激活抑制剂后,冻干前负载海藻糖过程对血小板的体外激活和聚集活性没有显著影响。

The aim of this research was to study the technology and methods of loading lyoprotectant-trehalose into cytoplasm of human platelets before lyophilization, to optimize experimental conditions of loading trehalose, to investigate the changes of platelets response to agonists and activation after incubation of platelets for 4 hours at 37℃ in the presence of lyoprotectant-trehalose, to protract the figures of loading efficiency and intracellular trehalose concentration versus incubation time, temperature and external trehalose concentration, to optimize loading parameters. The response of platelets to different agonists -- thrombin , ADP, collagen and ristocetin were measured respectively by APACT2 aggregometer before and after loading trehalose into platelets; the expressions of CD62p and PAC-1 on platelet membranes in the presence and absence of reversible platelets activation inhibitors were measured by flow cytometry respectively before and after loading trehalose into cytoplasm of platelets. The results showed that the loading efficiency was linear to incubation time （ 2 hours later） and incubation temperature （ rang from 30℃ to 40℃）, respectively. The loading efficiency almost reached 60% when the platelets were incubated at 37℃ for 4 hours. The intracellular trehalose concentration was higher with the increase of the extracellular trehalose concentration （ 〈 50 mmol/L ） . Compared to untreated groups, the values of MPV and aggregation to different agonists in treated groups showed no significant difference, respectively （ P 〉 0.01 ）. After incubation of platelets for 4 hours, the expression of CD62p increased to some extent, however, the expression of CD62p decreased again when the reversible platelets activation inhibitor PGE-1 and adenosine were added to the incubation buffer. It is concluded that 37℃, 4 hours and the extracellular trehalose concentration 〈 50 mmoL/L axe the optimal conditions for loading with trehalose. The processing of loading with trehalose before olatelet lyoohilization has no significant effects on response of platelets to agonists and activation.