小鼠冰冻血小板制备方法的建立及其质量评价

Establishment of a method for preparing cryopreserved platelets of mice and evaluating their quality

目的建立小鼠冰冻血小板(CPP)制备方法,并对制备的小鼠CPP进行质量评价。方法采用心脏采血方式获取小鼠全血后,用14%CPDA-1抗凝并分装入无菌离心管中,以离心力300×g、离心时间8 min的条件提取小鼠富血小板血浆(PRP),对PRP提取时的适宜离心体积进行探索以提高血小板回收率。采用Valeri“无清洗”方法对小鼠血小板进行-80℃冰冻储存,通过血小板计数、流式细胞分析、血小板聚集等评价CPP质量。结果全血离心体积为2.0 ml时血小板回收率最高,可达40%左右,且全血分层良好。相比新鲜血小板(FP),CPP冻存48 h后,血小板计数(PLT)无明显改变,血小板压积(PCT)、平均血小板体积(MPV)、血小板分布宽度(PDW)升高;血小板活化水平及凋亡率显著升高;血小板聚集率下降至40%左右。结论成功建立小鼠CPP制备方法,且制备的小鼠CPP质量良好,其测定指标的变化趋势与人源CPP相应指标的变化趋势相符。

Objective To establish a preparation method for and evaluate its quality of cryopreserved platelets(CPP)of mice.Methods After the whole blood of mice was obtained by cardiac blood collection,it was anticoagulated with 14%CPDA-1 and divided into sterile centrifuge tubes.Then,platelet rich plasma(PRP)was extracted with a centrifugal force of 300×g and centrifugation time of 8 min.Furthermore,the appropriate centrifugation volume of whole blood was explored to improve the platelet recovery rate.The mouse platelets were frozen and stored at-80℃using the method of“no washing after cryopreservation”.The quality of CPP was evaluated by platelet count,flow cytometry and platelet aggregation.Results The platelet recovery rate was the highest when the whole blood centrifuge volume was 2.0 ml,which was about 40%,and the whole blood was well stratified.Compared with fresh platelets(FP),after CPP cryopreservation of 48 hours,there was no significant difference in platelet count(PLT),but the mean platelet volume(MPV),platelet distribution width(PDW),and plateletcrit(PCT)increased.Platelet activation levels and apoptosis rate were significantly increased.The CPP aggregation rate decreased to about 40%.Conclusion A preparation method of mouse CPP is established,and the prepared mouse CPP is of good quality.The measurement indexes tend to change consistent with those of human CPP.