明胶-Ficoll法分离脐带血单个核细胞的方法学研究

Study on the methodology of mononudear cells isolation from umbilic cord blood by gelatin-Ficoll

目的比较直接利用Ficoll一步法和明胶-Ficoll两步法分离脐带血MNCs的效果。方法同一份脐带血分为两份,一份采用Ficoll直接分离MNCs,另一份先采用明胶沉淀红细胞后,再用Ficoll分离MNCs,比较两者的分离效率、细胞活力、细胞联苯胺染色阳性率、CD36和CD235a表达规律,以及CFU-E、BFU-E集落数。结果一步法和二步法的分离效率及细胞活力无统计学差异;一步法收集的细胞联苯胺染色阳性率高于二步法;一步法和二步法分离所得的MNCs表达CD36+/CD235a-和CD36+/CD235a+的细胞数无显著差异,但前者表达CD36-/CD235a+的细胞数比后者多;培养7天时CFU-E集落数,二步法稍低于一步法,14天时BFU-E集落数,二步法显著多于一步法。结论采用明胶-Ficoll二步法分离脐带血既不会增加MNCs的损失,还可更好地保留脐带血中比较早期的红系祖细胞。

Objective To compare the isolating efficiency of MNCs from umbilic cord blood（ UCB ） by gelatin-Ficoll and Ficoll alone. Methods The UCB was divided into two portions, one was isolated MNCs by Ficoll alone （one-step isolatin）, another was sedimented erythrocyte firstly, then isolated MNCs by Ficoll（two-step isolatin）. Results There was no significant statistic difference in the MNCs separating efficiency and cellular activity between the two-step and the one-step isolatin; The percentage of biphenylamine-positive cells is higher by the one-step isolation than two-step isolation; The number of cells expression CD36^＋/CD235a^- and CD36^＋/CD235a^＋ are similar compared the one-step with the two-step isolation, however, the number of CD36^-/CD235a^＋ cells are higher by the one-step than by the two-step method; The CFU-E colonies are a little lower at day 7 by the two-step than one-step method, while the BFU-E colonies are markedly higher at day 14 by the two-step than one-step method. Conclusion The two-step isolation using gelation-Ficoll will not make loss of MNCs from UCB, and it may keep the earlier erythoid progenitor cells.