骨髓造血干细胞分离 保存的研究

目的探讨骨髓造血干细胞分离及保存的方法。方法应用羟乙基淀粉(HES)或percoll液分离骨髓造血干细胞;联合应用二甲基亚砜(DMSO)和HES对造血干细胞进行液氮保存。应用血细胞计数法、锥虫蓝拒染实验、粒-巨噬细胞集落生成单位(CFU-GM)的体外培养等方法对造血干细胞冷冻前后的有核细胞(NC)数、存活率、体外分化能力进行检测;应用流式分析法计数CD34+细胞数。结果利用HES沉降法分离的单个核细胞数、CD34+细胞数、CFU-GM集落数均比percoll液离心法明显增多;骨髓造血干细胞冷冻保存1年后的有核细胞数、CD34+细胞数、锥虫蓝活率、CFU-GM集落计数与保存前差异无统计学意义。结论HES法分离骨髓造血干细胞方法安全、有效;通过程序降温,联合使用DMSO及HES的低温冻存方法对骨髓造血干细胞的长期保存是适合的。

Study on the separation and preservation of bone marrow hematopoietic stem cells

Objective To study the method of separation and preservation of bone marrow hematopoietic stem cells.Methods We separated bone marrow hematopoietic stem cells with hydroxyethyl starch(HES) and preserved them in liquid nitrogen by the use of dimethyl sulphoxide(DMSO) and HES,counted nucleated cells(NC) by blood cell counting chamber,detected cell surviving rate by trypan blue staining and evaluated stem cells differentiation ability by colony forming unit-granulocyte/macrophage(CFU-GM).The number of CD34 cells is counted by flow cytometry.The results showed numbers of mononucleated cells,CD34 cells and CFU-GM from HES sedimentation are marked increased than those from percoll solution centrifugation.Results There is no significant difference in numbers of nucleated cells,CD34 ,CFU-GM and viability rate after cryopreservation as compared with before cryopreservation.In conclusion,HES sedimentation is safe and effective for the separation of bone marrow hematopoietic stem cells.Conclusion Cryopreservation in liquid nitrogen by the use of DMSO and HES is an effective method for long-term preservation of hematopoietic stem cells.