人血小板冻干前负载海藻糖技术的研究论文.docVIP

　　人血小板冻干前负载海藻糖技术的研究论文 .freelmol/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50 mmol/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。 【关键词】 海藻糖 Process of Human Platelets Loaded of this research ethods 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 easured 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 easured by floetry respectively before and after loading trehalose into cytoplasm of platelets. The results shoe (2 hours later) and incubation temperature (rang from 30℃ to 40℃)，respectively. The loading efficiency almost reached 60% mol/L﹚. pared to untreated groups，the values of MPV and aggregation to different agonists in treated groups shoe extent，homol/L are the optimal conditions for loading mol/L NaCl、 10 mmol/L KCl、 10 mmol/L EGTA、 10 mmol/L咪唑、10 μmol/l 前列腺素-E1，pH 6.8)、80%甲醇溶液、硫酸蒽酮试剂、4种血小板聚集反应诱导剂： 凝血酶、ADP、胶原、瑞斯托霉素，荧光标记抗血小板表面糖蛋白单克隆抗体（2-8℃保存）：CD62p-PE、CD61-Percp、PAC-1-FITC、RGDS(PAC－1阻断剂)，含1% 多聚甲醛及0.1%叠氮钠的PBS（pH 7.2）固定液，可逆性血小板激活抑制剂PGE-1和腺苷（Sigma公司产品）。酶标仪、恒温水浴箱、抽真空干燥器、可调温度孵育箱、超声清洗器、分析天平，Nihonkohden(MEK-6108K)血细胞自动分析仪。兰波APACT2血小板聚集仪（美生实业有限公司），FACS流式细胞仪（美国Becton Dickinson公司），一次性12mm×75mm Falcon试管（Becton Dickinson公司）、CaliBRITE Beads/FACSp软件（FACS仪器校正，预设获