人胚AGM区造血干／祖细胞的分离及其在含AGM区基质细胞体系中的培养  

Isolation and Culture of Human Embryonic AGM Derived HSPCs in Hematopoietic Culture Systems Created by AGM Stromal Cells

作　　者：吴北燕[1] 黄绍良[2] 陈惠芹[2] 张绪超[3] 

机构地区：[1]汕头大学医学院第一附属医院儿科,广东汕头515041 [2]中山大学附属第二医院干细胞研究中心,广东广州510120 [3]广东省人民医院医学研究中心,广东广州510310

出　　处：《中国实验血液学杂志》2008年第3期579-583,共5页Journal of Experimental Hematology

基　　金：“十五”863计划资助项目(编号2003AA205008);广东省自然科学基金资助项目(编号7301460);广东省卫生厅医学科研基金课题(编号A2007423);汕头市重点科技计划项目(编号20068222)

摘　　要：本研究分离人胚主动脉-性腺-中肾(AGM)区造血干/祖细胞(HSPC)并在含人AGM区基质细胞的培养体系中进行培养,以了解人AGM区基质细胞对HSPC的作用。以免疫组织化学方法检测人胎龄28-45天胚胎AGM区细胞CD34、Flk-1、VEGF的表达;分离AGM来源的HSPC,通过直接接种和经Transwell非直接接触两种方法接种于含人AGM区基质细胞系(hAGMS3和hAGMS4)饲养层的培养体系,观察HSPC生长情况和卵石区形成细胞(cobblestone area-forming cells,CAFC)并计数卵石区(cobblastone area,CA);用间接免疫荧光方法检测培养体系中悬浮细胞和CAFC的CD34、Flk-1表达;收获细胞进行半固体造血集落培养以检测两种培养方法对AGM-HSPC的集落形成能力影响。结果表明:人AGM区造血细胞表达CD34和Flk-1。两种接种方法均显示有CFC形成,直接接触培养能形成CD34和Flk-1双阳性的CAFC,集落总数明显高于非接触培养(hAGMS3体系:1647±194vs389±31,p<0.05;hAGMS4体系:1586±75vs432±35,p<0.05)。结论:①人胚AGM区含有CD34+Flk-1+的HSC。②首次建立含人胚AGM基质细胞的AGM-HSPC培养体系,直接接触培养和非直接接触培养均能促进AGM-HSC的生长,AGM基质细胞与HSC直接接触发挥重要作用。This study was purposed to isolate human embryonic AGM derived HSPCs and investigate the effect of AGM stromal cells on AGM-derived HSPCs. Immunohistochemical sections of human AGM tissue were investigated for CD34, Flk-1 and VEGF expression. Human AGM-derived single cells were isolated and seeded onto pre-treated feeder of human AGM stromal cells （hAGMS3 and hAGMS4） by direct contact and non-contact co-culture in Transwell culture system. Growth characteristics of HSPCs with cobblestone area-forming cells （CAFCs） were observed and number of cobblestone area （CA） was counted. Indirect immunofluorescent assay was used to detect CD34 and Flk-1 expression on the surface of suspended cells as well as CAFCs in contact co-culture system. The cells after culture for 2 weeks were collected from both contact and non-contact co-culture systems for CFU assay. The result showed that hematopoietic cells in AGM tissue expressed CD34 and Flk-1. Both of the hematopoietic culture systems could produce CFCs. Nevertheless, direct contact co-culture produced CD34^＋ Flk-1 ^＋ CAFC and more CFUs than those from indirect non-contact culture （ hAGMS3 system： 1647 ± 194 vs 389 ± 31 ,p 〈 0.05 ; hAGMS4 system： 1586 ± 75 vs 432 ± 35, p 〈 0.05 ）. It is concluded that there were CD34 ^＋ Flk-1 ^＋ HSCs in human embryonic AGM region. The hematopoietic co-culture systems composed of AGM-derived HSPCs and AGM stromal cells are successfully established, both direct contact and Transwell non-contact co-culture can expand AGM-derived definitive HSPCs. Cell-cell contact between AGM-derived HSPCs and AGM stromal cells are of most importance to maintain and expand AGM-HSPCs.

关 键 词：永久造血 主动脉-性腺-中肾区 造血干细胞 基质细胞 卵石区形成细胞 

分 类 号：R318.04[医药卫生—生物医学工程] R322.2[医药卫生—基础医学]