不加羟乙基淀粉(HES)分离脐血造血干细胞的方法探讨

Discussion on the method of separating cord blood hematopoietic stem cells without adding hydroxyethyl starch(HES)

目的探讨在不添加羟乙基淀粉(HES)的情况下,分离获得脐血中造血干细胞的可行性。方法取健康产妇自愿捐献的脐血33份,每份均采用添加羟乙基淀粉(HES)和不添加羟乙基淀粉(HES)两种方法分离造血干细胞,计算有核细胞(TNC)回收率。所得干细胞成品,经程控降温后,保存于液氮中(-196℃),1个月之后取出成品进行复苏,采用台盼蓝拒染法检测细胞活力,流式细胞法结合7-AAD检测CD34+比值和活CD34+的比值,全自动血液分析仪计数分析TNC回收率,半固体培养法计数其粒-巨噬细胞集落形成单位数(CFU-GM)。结果两种方法在细胞活性、CFU-GM、制备TNC回收率、冷冻复苏TNC回收率和CD34+比值差异无统计学意义(P>0.05),活CD34+比值差异有统计学意义(P<0.05)。结论在不添加羟乙基淀粉(HES)情况下,通过提高离心力,能够很好的从脐血中分离质量指标可靠的造血干细胞。

Objective To explore the feasibility of separating hematopoietic stem cells from cord blood without adding hydroxyethyl starch(HES).Methods Took 33 umbilical cord blood voluntarily donated by healthy mothers and each portion was separated by two methods of adding hydroxyethyl starch(HES)and without adding hydroxyethyl starch(HES),finally the TNC recovery rate was calculated.The obtained stem cell products were stored in liquid nitrogen(-196℃)after programmed freezing,and the products were taken out for resuscitation after 1 month.The cell viability was detected by trypan blue rejection method,and the CD34+ratio and alive CD34+ratio were detected by flow cytometry combined with 7-AAD.TNC recovery rate was detected by automatic blood analyzer,and the number of granulocyte-macrophage colony forming units(CFU-GM)was counted by semi-solid culture method.Results There were no significant difference between the two methods in cell viability,CFU-GM,TNC preparation recovery rate,TNC cryo-resuscitation recovery rate and CD34+ratio(P>0.05),there was a significant difference in alive CD34+ratio(P<0.05).Conclusion Without adding hydroxyethyl starch(HES),by increasing the centrifugal force,hematopoietic stem cells with reliable quality index can be separated from cord blood.