血小板膜微粒的制备及止血功能的研究

Preparation of Platelet Membrane Microparticles and Their Hemostatic Function

目的探讨血小板膜微粒(IPMs)的制备工艺,并对研制的IPMs的止血功能及对凝血系统的影响进行动物实验研究。方法用街头采集的全血制备浓缩血小板悬液,用血小板型去白细胞滤器去除白细胞,轻离心去除红细胞后,用生理盐水洗涤血小板并调血小板浓度为2×109/ml,置-80℃反复冻溶3次,再用生理盐水洗涤,然后置60℃、20h灭活病毒,用高压匀质机进行匀质化破碎血小板膜,即为IPMs。用粒度测定仪检测IPMs的粒度;应用活化血浆凝固时间(APCT)检测IPMs的体外促凝血活性;将IPMs输入血小板减少症兔出血动物模型体内,观察IPMs止血效果及对凝血系统的影响。结果制备的IPMs颗粒均匀,大小为200～300nm;50μg/mlIPMs相当于250×109/L新鲜血小板的体外促凝血活性;将IPMs按2mg/Kg输入兔血小板减少症的出血动物模型体内,在输注2～12h内,兔耳出血时间和APCT均明显缩短,而其他凝血指标(PT、APTT、Fg、TT)均无明显变化。结论血小板膜微粒止血效果好,输注IPMs并不影响凝血系统,是一个很有前途的生物止血剂。

Objective To investigate the preparation technique of infusible platelet membrane micropartieles （IPMs） ,and evaluate hemostatie funetion and effect of IPMs on pathologieal thrombogenieity after transfused into model of rabbit thrombocytopenia. Methods Whole bloods colleeted in the street were used to prepare platelet concentrates（PCs）, white cells were removed by white cell filter, and red cells by centrifugation at 1 000 r/min for 10 min. After that, platelets were washed twice with 0. 9% NaCl solution by repeated resuspension and eentrifugation,and adjusted concentration to 2 × 10^9/ml with 0. 9 % NaCl. The washed platelets were disrupted by repeated freezing （at - 80℃ ） and thawing （at 25℃ ） three times,and were heated at 60℃ for twenty hours to inactivate any viral contaminants. Finally,the heat-treated platelets were crushed by high pressure homogenizer to form IPMs. In this study, partiele analyser was applied to determine the diameter of IPMs,APCT to test the proeoagulation activity of IPMs in vitro. Hemostatic function and effect of IPMs on pathological thrombogenicity were observed after IPMs were transfused into thrombocytopenia rabbit models. Results Diameter of IPMs was from 200 to 300 nm. The proeoagulatiov activity of 50μg/ml of IPMs was equal to 250× 10^9/L of fresh platelets. Rabbit ear bleeding time and APCT significantly shortened from two hours to twelve hours after transfusion of 2 mg IPMs per Kg into thrombocytopenia rabbit models,but other indexes such as PT,APTT,Fg,and TT changed less. Conelusion IPMs possess good hemostatic function and less effect on pathological thrombogenicity. It is a kind of promising biologic hemostatie reagent.