S-亚硝基谷胱甘肽抑制血小板凋亡的体外研究

Anti-apoptotic effect of s-nitrosoglutathione on platelet in vitro

目的探讨一氧化氮(NO)供体S-亚硝基谷胱甘肽(GSNO)对储存期内机采血小板细胞凋亡的影响。方法 6份机采血小板,每份分装到2个血小板保存袋,随机分到实验组和对照组,实验组加入GSNO(终浓度为100μmol/L),对照组加入无菌生理盐水。于(22±2)℃震荡保存,分别于第1、3、5、7天取血小板进行扫描电镜观察,采用流式细胞仪检测血小板凋亡情况,采用蛋白免疫印迹法检测凋亡相关蛋白表达。结果实验组血小板NO含量高于对照组(P<0.05)。血小板扫描电镜图显示,实验组血小板显微形态在外形、减少聚集等方面均优于对照组。在血小板保存过程中,乳酸含量不断累积(P<0.05),葡萄糖含量不断消耗(P<0.05),PS膜外翻的血小板数量不断增加(P<0.05);且实验组血小板乳酸、葡萄糖含量和PS膜外翻阳性率均低于对照组(P<0.05);凋亡相关蛋白Bax、Bcl-x L和Bak表达水平均未随保存时间延长而发生显著变化(P>0.05),两组上述三种蛋白表达水平差异均无统计学意义(P>0.05)。结论在血小板保存过程中加入NO供体GSNO,可在一定程度上抑制血小板细胞凋亡,减少血小板贮存损伤。

Objective To investigate the effects of nitric oxide（NO） donor s-nitrosoglutathione（GSNO） on apoptosis of apheresis platelets cells during storage. Methods Six platelets were collected and divided into two bags aseptically. One bag for experimental group and the other for control group into which a GSNO solution with a certain concentration was added into fresh apheresis platelets. Scanning electron microscope, flow cytometry and Western Blot were used to detect platelet morphology,apoptosis markers and the expression of apoptosis-related protein,respectively. All the parameters were dynamically monitored at the 1 st,3 rd,5 th,and 7 th day. Results There was a difference of NO concentration between GSNO group and control group（P〈0.05）,the NO level in GSNO group were remarkably higher than those in the control group during the whole storage stage.Platelet scanning electron microscopy showed that the morphology of platelets in GSNO group was superior to that of the control group. There were differences in lactate acid content（P〈0.05） and glucose（P〈0.05） in GSNO group and the control group,and GSNO group was significantly lower than control group at some time point. Flow analysis showed the positive ratio of phosphatidyl serine（PS） exposed on the cell surfaces,and GSNO group was lower than control group（P〈0.05）. There was no statistical difference between the relative expression levels of apoptosis-related protein（P〉0.05）. Conclusion The addition of NO donor GSNO in platelet preservation can inhibit platelet cell apoptosis and reduce platelet storage lesion in vitro during the platelet storage period.