体外培养扩增的人脐血CD34+/细胞形成HPP-CFC的能力

目的：评价ＣＤ３４＾＋细胞体外扩增时高增殖潜能集落形成细胞（ＨＰＰ－ＣＦＣ）的变化。方法：利用ｍｉｎｉ－ＭＡＣＳ分离脐血ＣＤ３４＾＋细胞，流式细胞仪（ＦＡＣＳ）测定细胞纯度（８６％～９５％）。应用含ｒｈＳＣＦ、ｒｈＩＬ－３、ｒｈＩＬ－６和ｒｌＧＭ－ＣＳＦ的培养体系扩增ＣＤ３４＾＋细胞。分别于培养的第１，２和４周收取细胞进行ＨＨＰ－ＣＦＣ测定，测试体系为含重组人ＳＣＦ、ＧＭ－ＣＳＦ、ＩＬ－３、ＥＰＯ等细胞因子的半固体甲基纤维素。结果：测试体系中加入单个细胞因子不能产生ＨＰＰ－ＣＦＣ，含ｒｈＩＬ－３和ｒｈＧＭ－ＣＳＦ体系产生的集落小且主要为巨噬细胞集落，由所有因子组成的体系产生的集落大而致密，包含粒系３种类型。此外，尽管细胞总数量显著增加，扩增的ＣＤ３４＾＋细胞ＨＰＰ－ＣＦＣ形成率随培养时间的延长而减少。结论：本方

Ability of in vitro amplified CD34~ cells from human umbilical cord blood to form HPP-CFC

Objective:To evaluate the changes of colony forming cells with high proliferative potential (HPP CFC) in human CD34 cell culture after in vitro amplification.Methods:CD34 cells were obtained from umbilical cord blood by mini MACS immunomagnetic selection and the cell purity was 86 95% measured by flow cytometry analysis. The isolated cells were incubated for 4 weeks in the presence of rhSCF, rhIL 3, rhIL 6 and rhGM CSF. Cells harvested from the culture were set in methylcellulose with cytokine cocktail of rhSCF, rhGM CSF, rhIL 3, rhIL 6, and rhEPO.Results:Cultures with the individual cytokines tested could not give rise to HPP CFC. The colonies formed in the culture system containing rhIL 3 and rhGM CSF were small in diameter and mainly consisted of macrophages.Combination of all the tested cytokines, however, resulted in large and dense colonies containing the three myeloid cell types. Furthermore, the frequency of HPP CFC in CD34 cell culture decreased gradually with the culture time, although the absolute cell numbers increased dramatically. Conclusions:After in vitro amplification, the number of HPP CFC from CD34 cells was maintained as evaluated by the method described.