摘要
为了探索从胎盘组织中分离富集造血干/祖细胞(HSPC)的标化流程,采用机械法加胶原酶消化法制备人胎盘组织单个细胞悬液,用羟乙基淀粉(6%HES)法从中分离出单个核细胞(MNC),再经免疫磁珠分选法分选出CD34-、CD34+CD38-、CD34+CD38+3个细胞亚群,用流式细胞术对各阶段分选细胞进行表型分析并计算分选细胞的富集度和回收率。结果表明:机械法加胶原酶消化法制备的人胎盘组织单个细胞悬液中单个核细胞(MNC)数达(12.30±3.51)×108,与脐血初始样品所含的MNC数(8.86±5.38)×108比较差异无统计学意义,而其CD34+细胞所占百分率[(3.93±2.31)%]则明显高于脐血[(0.44±0.29)%]。胎盘组织单个细胞悬液经6%HES分离后MNC和CD34+细胞的回收率分别为(45.3±11.7)%和(51.1±9.8)%;MNC经免疫磁珠分选后,其CD34+细胞的纯度和回收率分别为(73.4±14.1)%和(52.7±11.7)%。结论:本实验所建立的"机械法加胶原酶消化法-HES分离MNC-MACS分选目标细胞"的分离纯化方法可从胎盘组织获得高丰度、高富集度、高活性的HSPC,为进一步研究胎盘HSPC提供了比较经济、效果较好的分离富集方案。
The aim of this study was to establish the standard protocols for isolating and enriching hematopoietic stem/progenitor cells （HSPC） from human placenta tissue （PT）. Single-cell suspension from of human PT was prepared by mechanical method combined with collagenase digestion. Mononucleated cells （MNC） derived from PT were separated by hydroxyethyl starch （ 6% HES ）, then the three cell subsets of different immunophenotypes （ CD34^-, CD34^＋ CD38^ -, CD34^＋ CD38^＋ ） contained in MNC were isolated by Magnetic Activated Cell Sorting （ MACS ）. The cell immunophenotype of each sorting steps was analyzed by flow cytometer （FCM）. The cell enrichment and recovery rate of each sorting step were calculated. The results showed that MNC could be harvested up to （ 12.30 ±3.51 ） × 10^8 from a single-cell suspension of human PT by mechanical method and collagenase digestion, no significant difference existed as compared with umbilical cord blood （ UCB ） initial sample [ （ 8.86 ± 5.38 ） × 10^8 ], but the percentage of CD34^＋ cells in MNC of human PT was （3.93 ± 2.31 ） %, much higher than that in UCB [ （ 0.44 ± 0.29 ） % ] （ P 〈 0.001）. recovery rate of MNC and CD34^＋ cells from PT after separation with 6% HES were （45.3 ± 11.7）% and （ 51.1 ± 9.8 ） % , respectively. After MNC being sorted by MACS, the enrichment and recovery rate of CD34 ^＋ cells in CD34^＋ group were （73.4 ± 14.1 ） % and （ 52.7 ± 11.7 ） % respectively. It is concluded that the protocols established here for isolating and enriching hematopoietic stem/progenitor cells from human placenta can acquire HSPC with high abundance, enrichment and viability and may be a useful reference of isolating methods for future related study.
出处
《中国实验血液学杂志》
CAS
CSCD
2006年第5期955-958,共4页
Journal of Experimental Hematology
基金
贵州省科技计划资助项目(黔科合计字[2003]JGY005)
