【作者】 卢发强；

【导师】 刘景汉；

【作者基本信息】 中国人民解放军军医进修学院 ， 临床检验诊断学， 2006， 博士

【摘要】 目的 研究人血小板冻干前负载海藻糖技术与方法,筛选合适负载条件,可逆性抑制血小板激活,保护血小板功能。并以此检测血小板冻干再水化后特性。 方法 应用硫酸-蒽酮反应,定量检测载入到血小板胞内的海藻糖,绘制血小板胞内海藻糖浓度及负载效率随温度、时间、胞外海藻糖浓度变化曲线,制定出合适负载海藻糖条件。使用APACT-2型血小板聚集仪,分别检测血小板负载海藻糖前后聚集反应性变化。使用流式细胞仪分别测定血小板在自身血浆和负载缓冲液中负载海藻糖前后、在含有和未含有DMSO（2%）溶液的缓冲液中负载海藻糖后,血小板膜表面糖蛋白CD_62P、PAC-1的表达。应用硫酸-蒽酮反应,测定血小板在含有和未含有DMSO（2%）缓冲液中,37℃条件下,负载海藻糖4h后的胞内海藻糖浓度。用激光共聚焦荧光显微镜观察分析荧光黄CH（LYCH）被负载入血小板内的量及分布随DMSO（2%）的添加、负载温度、及负载时间变化的趋势。分别使用流式细胞仪和硫酸-蒽酮反应,测定分析不同浓度可逆性激活抑制剂PGE₁和腺苷添加后,血小板膜表面CD_62P、PAC-1的表达和血小板负载海藻糖效率。用血细胞计数仪检测再水化冻干血小板计数回收率随冻干液中胞外海藻糖、人血清白蛋白浓度、及室温放置时间的变化趋势。使用APACT-2型血小板聚集仪分别检测新鲜和再水化冻干血小板对四种不同浓度诱导剂的聚集反应,检测冻干液中胞内外海藻糖浓度对冻干再水化血小板聚集反应的影响。使用流式细胞仪分别检测分析胞内、外海藻糖浓度及低浓度DMSO溶液的添加对再水化冻干血小板膜表面CD_62P、PAC-1表达的影响。用全自动血凝仪检测富含血小板血浆（PRP）冻干再水化后不稳定凝血因子Ⅷ和V活性变化。结果 （1）建立了血小板胞内海藻糖提取和定量测定方法。37℃时,4h,胞外海藻糖浓度<50mmol/L为冻干前血小板负载海藻糖合适条件。（2）血小板负载海藻糖前后聚集反应性无显著性差别（P>0.05）。在含有可逆性激活抑制剂腺苷（5mmol/L）和PGE₁（10μg/ml）的缓冲液中负载海藻糖更多还原

【Abstract】 Objective The aim was to study the technology of loading trehalose into cytoplasm of platelets before lyophilization, to optimize conditions for loading trehalose, to inhibit reversibly platelets activation, and to study the characteristics of rehydrated and lyophilized human platelets. Methods The sulfuric-anthrone reaction was used to determine the intracellular trehalose concentration loaded by human platelets.The figures of loading efficiency or intracellular trehalose concentration versus incubation temperature, time, and external trehalose concentration were protracted respectively to optimize loading conditions. The responses of platelets to agonists were assayed by APACT-2 aggregometer before and after platelets incubation. The expressions of CD62_P and PAC-1 on platelets membranes were measured by flow cytometry （FCMs） before and after incubation of platelets in plasma or loading buffer at 37℃ for 4h_s, in the presence and absence of 2% DMSO in loading buffer, respectively. The sulfuric-anthrone reaction was used to measure the intracellular trehalose concentrations after incubation of platelets at 37℃ for 4h_s in loading buffer, in the presence and absence of 2% DMSO, respectively. The intracellular LYCH concentration and distribution versus incubation temperature, and time, were visualized respectively by fluorescence microscopy in the presence and absence of 2% DMSO. The expressions of CD_62P and PAC-1 on platelets membranes, and intracellular trehalose concentration were measured by FCMs and sulfuric-anthrone reaction respectively, in the presence and absence of reversible platelets activation inhibitors-Adenosine and Prostaglandin E₁, before and after incubation of platelets at 37℃ for 4h_s. The effects of external trehalose or human serum albumin concentration added to drying buffer, and storage time at room temperature on numerical recoveries of rehydrated-lyophilized platelets were analyzedrespectively by counting platelets with a Blood Cell counter MEK-6108K. The dose-responses of rehydrated and fresh platelets to agonists and the effects of intracellular or external trehalose concentration on aggregation were measured respectively by APACT-2 aggregometers. The effects of intracellular or external trehalose concentration and 2% DMSO on activation of （p）rehydrated platelets were analyzed respectively by FCMs. The Hemostasis Analyzer was used to measure the activities of unstable coagulation factorⅧ and factor V in plasma before and after platelet-rich plasma were lyophilized and rehydrated. Results （1） The method for intracellular trehalose concentration was established. Incubation temperature of 37 degrees C, incubation time of 4 h_s, and external trehalose concentration < 50mmol/L in loading buffer were the optimal conditions for platelets loaded with trehalose before lyophilization. （2） Compared to untreated groups（before incubation in plasma）, the responses of platelets to agonists in treated groups （after incubation in plasma） showed no significant difference, P>0.05;The expression of CD_62P was higher after incubation of platelets in loading buffer for 4 h_s, P<0.01, and decreased again with the reversible platelets activation inhibitors-Adenosine （ 5mmol/L ） and Prostaglandin E₁ （PGE₁,10 μ g/ml） added to loading buffer, P>0.05;The expressions of PAC-1 kept relatively stable. Compared to untreated groups（before incubation in plasma）, the expressions of CD_62P and PAC-1 showed no significant difference after incubation of platelets in plasma at 37℃ for 4h_s, P>0.05. （3） Compared to controlled groups （loading in buffer）, the expressions of CD62_P and PAC-1 in treated groups （loading in plasma） decreased significantly, （8.29 + 8.69） % versus （20.67 + 7.557） %, respectively, P<0.01. （4） Compared to controlled groups （loading in buffer in the absence of 2% DMSO）, the expressions of CD_62P and the intracellular trehalose concentration in treated groups （loading in buffer in the presence of 2% DMSO） decreased significantly, P<0.01;The intracellular LYCH concentration was higher, and homogeneously distributed in the cytoplasm of platelets. （5） Reversible platelets activation inhibitors-Adenosine （5 mmol/L） and PGE₁ （10 μ g/ml） significantly inhibited the expressions of glycoprotein CD_62P and PAC-1, and significantly increased the loading efficiency of trehalose. （6） The numerical recoveries of lyophilized and rehydrated platelets loaded with trehalosevaried with external trehalose or human albumin concentration in freeze-drying buffer, and were much more stable after rehydration of lyophilized platelets at room temperature for 8 h_s. （7） The dose-response curves of rehydrated platelets to agonists were similar to that of fresh platelets. Compared to untreated groups （without loading trehalose before lyophilization, only in presence of external trehalose in drying buffer）, the values of aggregation of rehydrated platelets to ADP, and the expressions of glycoprotein CD_62p in the treated groups （loading trehalose before lyophilization, in the presence of external trehalose in the freeze-drying buffer） showed significant difference, respectively, P<0.01. The expressions of CD_62P decreased significantly with 2% DMSO, Adenosine or/and PGE₁ added to drying buffer, P<0.01, respectively. （8） The activities of unstable coagulation factor Ⅷ and V before and after lyophilization showed no significant differences in the presence of trehalose in drying buffer, P>0.05. Conclusions Incubation temperature of 37 degrees C, incubation time of 4 h_s, and external trehalose concentration <50mmol/L in loading buffer were the optimal conditions for platelets loading trehalose. Loading trehalose in plasma more effectively inhibited platelets activation than in loading buffer. Reversible platelets activation inhibitors-adenosine （5mmol/L） and PGE₁ （10μg/ml） significantly inhibited platelets activation. DMS0（2%） significantly accelerated the intake of trehalose and made trehalose homogeneously distributed in cytoplasm of platelets, and effectively inhibited platelets activation in all processing of platelets lyophilization and rehydration. The numerical recoveries of lyophilized and rehydrated platelets were higher and much more stable at room temperature. The rehydrated-lyophilized platelets had good responses to agonists. The activities of unstable coagulation factor Ⅷ and V in PRP kept stable before and after lyophilization in the presence of trehalose.更多还原