人胎盘CD133^＋细胞具有高增殖潜能集落形成细胞特性

PROPERTIES OF HPP-CFC OF CD133^+ CELLS DERIVED FROM HUMAN PLACENTA

目的通过对人胎盘CD133+细胞群中高增殖潜能集落形成细胞(HPP-CFC)检测与生物学特性的分析,证明人胎盘存在早期造血干/祖细胞(HSPC)。方法采用机械法制备人胎盘组织(PT)单细胞悬液,用Histopaque-1007分离出单个核细胞(MNC),经磁式分选(MACS)富集CD133+细胞,培养28d后观察HPP-CFC集落形成能力,用流式细胞仪(FCM)对分选的细胞组份和HPP-CFC进行表型分析,实验全程用脐带血(UCB)作平行比较分析。结果培养28d后,PT-CD133+与UCB-CD133+细胞组份分别扩增了266和362倍,前者低于后者(P<0.01);PT-CD133+与UCB-CD133+细胞中HPP-CFC分别为(32.4±11.2)/5×103、(17.7±5.7)/5×103,前者形成的HPP-CFC数量明显高于后者(P<0.01);PT-CD133+、UCB-CD133+细胞培养至28d时,除UCB-CD133+组的CD133+CD34-亚群比例无明显改变外,CD133+CD34+、CD133-CD34+和CD133+CD34-(PT-CD133+组)亚型均比培养前减少。结论人胎盘组织CD133+细胞中存在HPP-CFC,说明胎盘CD133+细胞群中存在早期HSPC。

Objective To investigate whether human placenta tissue （PT） CD133^＋ cells possess the high proliferative potential colony forming cells （HPP-CFC） and analyze its properties in order to verify that human PT contains primitive hematopoietic stem/progenitor cells（ HSPC）. Methods Single cell suspension liquid of human PT was prepared by mechanical method. After that the mononueler cells（MNC） from PT was separated by Histopaque-1007 agent. CD133^＋ cells contained in MNC were isolated and purified by magnetic activated cell sorting（MACS） method. After 28 days of HPP-CFC expansion culture of CD133^＋ cells, the frequency and morphology of HPP-CFC were counted and observed. Phenotypes of purified CD133^＋ ceils and HPP-CFC were analyzed by flow cytometer（FCM）. In parallel, human umbilical cord blood（UCB） samples underwent the same protocols for comparison. Results After 28 days of culture the expansion fold of PT-CD133^＋ cells was 266, lower than that of UCB-CD133^＋ cells which was 362 （ P 〈 0.01）. The number of HPP-CFC in CD133^＋ cells derived from human PT and UCB were （32.4 ±11.2）/5×10^3 and （17.7 ±5.7）/5 ×10^3 respectively, the number of the former obviously higher than that of the latter （P 〈 0.01）. FCM analysis results showed that, except for CD133^＋ CD34^- subset from UCB-CD133^＋ cells, the proportion of CD133^＋ CD34^＋ , CD133^- CD34^＋ subsets both from PT- and UCB-CD133^＋ cells and CD133^＋ CD34^- subset from PT-CD133^＋ cells were decreased through 4 weeks of culture. Conclusion Human PT-CD133^＋ cells can form HPP-CFC, suggesting that the cell population of human PT-CD133^＋ cells contain early HSPC.