非连续密度梯度离心法分离红细胞的影响因素研究  

Affected Factors of Separating Erythrocytes by Discontinuous of Density Gradient Centrifugation

作　　者：林梦霞[1] 于帅[2] 祝超[3] 黄雅 王海宝[1] 于洋[4] 

机构地区：[1]解放军总医院海南分院输血科,海南三亚572013 [2]解放军总医院海南分院检验中心,海南三亚572013 [3]海南医学院热带医学与检验医学院,海南海口571199 [4]解放军总医院输血科,北京100853

出　　处：《中国实验血液学杂志》2017年第3期904-910,共7页Journal of Experimental Hematology

基　　金：三亚市医疗卫生科技创新项目(2014YW30)

摘　　要：目的:研究非连续密度梯度分离法分离红细胞的影响因素,初步建立Percoll分离液在红细胞分离中的最佳操作条件。方法:检测Percoll与1.5 mol/L NaCl比例为9∶1、10∶1、11∶1、12∶1、13∶1渗透压情况,选取等渗比例作为最佳母液(SIP);以不同密度细胞被充分离心沉降分离,较少存在红细胞滞留、拖带现象作为分离效果评判标准,比较不同红细胞悬液浓度、不同离心力、不同离心加减速度、不同温度和不同离心时间的分离效果。结果:50%以下浓度红细胞悬液分离效果优于50%以上浓度红细胞悬液,4 000×g优于3 000和2 000×g的离心效果,2-8的离心加减速度可以获得满意的离心效果,4℃分层效果比室温稳定,20 min优于15和10 min的分离效果。结论:Percoll与1.5mol/L NaCl为12∶1和11∶1的母液可作为等渗母液,优先4℃离心条件离心,加减速度2-8,其离心效果会随着红细胞悬液浓度的降低、离心时间的延长。Objective： To study the affected factors of the separation of red blood cells by discontinuous density gradient method, and to establish the optimal operating conditions of percoll for the separation of erythrocytes. Methods ： The osmolality of different ratio of Percoll and l. 5 mol/L NaC1 were 9： 1, 10： 1, ll： 1, 12： 1, 13：1 ratio, was detected respectively. The isotonic osmolality ratio was selected as optimum stock isotonic Percoll solution （SIP）, Evaluation criteria： All the different density cells were separated sufficiently by centrifugation, there is little red cell retention and entrainment phenomena. The separation efficacy of different red cell suspension concentration, different centrifugal force, different centrifugal speed, different temperature and different time were compared. Results： The separation efficacy of red cell suspension concentration which not exceed 50% was better than above 50%, efficacy of 4 000 x g was superior to 3 000 x g and 2 000 x g, 2 - 8 centrifugal acceleration and deceleration centrifugal could obtain the satisfactory efficacy, the 4 ℃ layered effect was more stable than room temperature, the separation effect of 20 min was better than 15 min and 10 rain. Conclusion： Percoll and 1.5 mol/L NaC1 12：1 to 11：1 can be used as stock isotonic percoll solution, preferentially selected 4 ℃ as centrifugal conditions. When conditions of separation was satisfied, the centrifugal acceleration and deceleration is 2 - 8, the optimal centrifugal effect can be obtained with the decrease of erythrocyte suspension concentration, with centrifugation time prolonging and centrifugal force increasing.

关 键 词：非连续密度梯度离心 梯度分离 红细胞 

分 类 号：R331.141[医药卫生—人体生理学]