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造血发育过程中造血祖细胞生物学特性的时空变化

周琨, 黄丽芳, 胡采红, 刘文励, 孙汉英^*

华中科技大学同济医学院附属同济医院血液科，武汉 430030

摘要

本实验以小鼠胚胎主动脉- 性腺- 中肾(aorta-gonad-mesonephros, AGM)区、胎肝(fetal liver, FL)以及小鼠骨髓(bonemarrow, BM)为研究对象，研究各阶段造血祖细胞在发育过程中的c-kit 的表达变化和部位迁移，以及不同发育阶段、不同造血部位来源c-kit+ 细胞的生物学特性。用流式细胞术检测小鼠胚胎10.5 天(embryonic 10.5, E10.5)、E11.5 的AGM区，E12.5、E14.5、E16.5、E18 的FL 以及BM 来源的单个核细胞中c-kit+ 细胞的比例。用磁珠分选法分离E10.5 AGM 区、E14.5 FL以及BM 来源c-kit+ 细胞，贴壁培养法分离小鼠E14.5 FL 基质细胞。建立体外Transwell 共培养体系，比较不同来源的c-kit+ 细胞增殖、分化、迁移以及集落形成能力。流式细胞术检测发现:相对于BM，E10.5 AGM 区和E12.5 FL 中c-kit+细胞比例较高。进行体外培养，AGM 区来源c-kit+ 细胞具有较强的增殖潜能，而FL、BM 来源的c-kit+ 细胞在体外培养中更容易分化；AGM 区和FL 来源的c-kit+ 细胞比BM 来源的c-kit+ 细胞在体外培养中具有更强的趋化迁移能力。集落形成能力方面，AGM 区来源c-kit+ 细胞具有较强形成混合集落形成单位(CFU-Mix)的能力；FL、BM 来源c-kit+ 细胞则能形成更多粒单系集落形成单位(CFU-GM)。以上结果表明，和FL、BM 来源的c-kit+ 细胞相比，AGM 区来源c-kit+ 细胞具有更强的体外增殖、多系分化潜能以及趋化迁移能力，在未来可能有着更广阔的应用前景。

关键词： 造血祖细胞; 造血发育; 主动脉- 性腺- 中肾区; 胎肝; 骨髓

分类号：R331.1;R329.28;R322.2

[Biological characteristics of hematopoietic progenitor cells at different stages ofhematopoietic development.] [Ariticle in Chinese]

ZHOU Kun, HUANG Li-Fang, HU Cai-Hong, LIU Wen-Li, SUN Han-Ying^*

Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

Abstract

The aim of the present paper is to better understand the mechanism of hematopoietic development through studying the biological characteristics of hematopoietic progenitor cells at different stages of development. Firstly, the c-kit expression levels of the mononuclear cells from murine embryonic aorta-gonad-mesonephros (AGM) region at embryonic day (E)10.5 and E11.5, fetal liver (FL) at E12.5, E14.5, E16.5, E18 and bone marrow (BM) were assayed with fluorescence activated cell sorting (FACS). Secondly, hematopoietic progenitor cells derived from AGM at E10.5, FL at E14.5 and BM were isolated by using c-kit microbeads. Isolated c-kit+ population cells from AGM, FL and BM were then co-cultured with E14.5 FL-derived stromal cells in transwell co-culture system in vitro. After 3, 7, 10 days of co-culture, numerous floating cells were generated. The floating cells generated in transwell inserts were collected for FACS cell count, migration activity detection and colony forming unit (CFU) formation assay. The results showed that the c-kit was highly expressed in E10.5 AGM, with the percentage of c-kit+ cells declining during AGM development. c-kit expression was highly expressed again in E12.5 FL, declining along with the progressive development of the FL region. Co-cultured with FL-derived stromal cells, E10.5 AGM-derived c-kit+ cells produced the highest number of hematopoietic cells, while BM-derived c-kit+ cells produced the lowest number of hematopoietic cells. Compared with E10.5 AGM-derived c-kit+ cells, E14.5 FL- and BMderived c-kit+ cells inclined to differentiate after 7 to 10 days of culture in vitro. E10.5 AGM and E14.5 FL-derived c-kit+ cells exhibited a higher migration activity than BM-derived c-kit+ cells. Moreover, E10.5 AGM-derived c-kit+ cells showed a higher ability to form mixed colony-forming unit (CFU-Mix) colony. In conclusion, compared with FL- and BM-derived c-kit+ cells, E10.5 AGM-derived ckit+ hematopoietic progenitor cells exhibit better proliferation, migration potential, and have a higher ability to maintain the undifferentiation state in vitro, providing an insight into their clinical manipulation.

Key words: hematopoietic progenitor cells; hematopoietic development; aorta-gonad-mesonephros; fetal liver; bone marrow

收稿日期：2009-05-31　　录用日期：2009-08-04

通讯作者：孙汉英　　E-mail: shanhum@163.com

引用本文：

周琨, 黄丽芳, 胡采红, 刘文励, 孙汉英. 造血发育过程中造血祖细胞生物学特性的时空变化[J]. 生理学报 2009; 61 (5): 493-498.

ZHOU Kun, HUANG Li-Fang, HU Cai-Hong, LIU Wen-Li, SUN Han-Ying. [Biological characteristics of hematopoietic progenitor cells at different stages ofhematopoietic development.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (5): 493-498 (in Chinese with English abstract).