二甲亚砜对血小板冻存中功能保护作用的实验研究

Protective Effects of DMSO on Function of Lyophilized Human Platelets

本研究探讨二甲亚砜(DMSO)对血小板冻干保存中激活的抑制作用。以CD62p和PAC-1表达作为血小板的激活标志,CD62p和PAC-1再表达和血小板最大聚集率作为评价血小板功能的指标。对血小板经离心、洗涤及海藻糖负载等预处理过程中加与不加DMSO的实验组,分别进行血小板膜表面糖蛋白CD62p和PAC-1表达及再表达的流式细胞术(FCMs)分析,血小板最大聚集率和血小板平均体积(MPV)的测定,研究DMSO对血小板激活抑制作用的可逆性。结果显示,血小板经预处理后,不含DMSO组的CD62p和PAC-1表达率显著增高,分别达30.37%和15.01%;含DMSO的组,CD62p表达率为10.72%,PAC-1几乎不表达(0.11%),显著低于不含DMSO组(p<0.01)。DMSO稀释后,血小板CD62p再表达为54.39%,显著低于对照组(71.69%)和高于不含DMSO组(35.98%)(p均<0.01);PAC-1再表达率为49.28%,与对照组(54.48%)无显著差异,显著高于不含DMSO组(p<0.01)。在含DMSO组,血小板用原血浆稀释后,血小板聚集功能恢复,对restocetin,thrombin和propylgal late诱导的血小板聚集率分别为92.76%,91.24%和89.66%,与对照组和无DMSO组比无显著性差异,对ADP诱导的聚集率有34.33%,显著高于无DMSO组(p<0.01),但仍显著低于对照组(p<0.01)。结论:DMSO对体外处理所致的血小板膜表面CD62p和PAC-1表达具有抑制作用,且该抑制作用是可逆的,DMSO稀释后血小板仍具有CD62p和PAC-1表达能力和聚集反应功能,提示DMSO具有抑制激活损伤和低温保护的双重功能,这一生物学特性使DMSO在血小板的冻干保存中发挥重要作用。

This study was aimed to investigate the effects of DMSO on platelets during pre-treatment for lyophilization, including centrifugation, washing and loading trehalose. After pre-treatment for lyophilization, the expression of platelet membrane surface glycoprotein （GP） including CD62p and PAC-1 was analyzed by FCM before and after induction with thrombin, the mean platelet volume （MPV） and platelet maximal aggregation with several platelet inducers were investigated. The results showed that the expression rates of CD62p and PAC-1, as the platelet activation signs, increased and were 30.37% and 15.01% respectively in group without DMSO after pre-treatment. And their differences in comparison with control were statistically significant, but that of CD62p was 10.72% and PAC was 10. 11% in group with DMSO, in comparison with group without DMSO respectively, their differences were statistically significant after diluting with DMSO, CD62p was re-expressed to 54.39% in group with DMSO and more than that in group without DMSO and lower than control statistically significant. PAC-1 was re-expressed to 49.28% in group with DMSO and more than that in group without DMSO （p 〈 0. 01 ） and reached to control. Platelet maximal aggregations induced by thrombin, restocetin and propyl gallate were 92. 76%, 91.24% and 89.66 respectively in group with DMSO. These were closed to that in control group and in group without DMSO. But the aggregation induced by ADP was 34.33 %, it was less than control （p 〈0.01 ） and more than that in group without DMSO （p 〈0.01 ）. It is concluded that DMSO can inhibit the expression of CD62p and PAC-1 on platelet in vitro. But when diluted with plasma, platelets can express CD62p and PAC-1 induced by thrombin and be led to aggregate by several inducers, so the inhibitory effects of DMSO on platelet activation are reversible. DMSO play roles in inhibitor damage from platelet activation and cryoprotectant.This property of DMSO is very important in research of platelets lyophilization.