拟胚体培养体系中造血表面标志的变化及其向红细胞分化潜能的研究  

A study on the transition of hematopoiesis surface marker and erythroid differentiation potential using EB culture system

作　　者：路旭琳 郁金凤 孙文翠[1] 毛斌[1] 邹庆 潘旭[1] 赖默温 周涯[1] 李晴[1] 曾宪波[1] 黄淑[1] 周琼秀[1] 周家喜[3] 马峰[1,3,2] 

机构地区：[1]中国医学科学院北京协和医学院输血研究所,四川成都610052 [2]四川新生命干细胞科技股份有限公司 [3]中国医学科学院北京协和医学院血液学研究所实验血液学国家重点实验室

出　　处：《中国输血杂志》2015年第5期488-492,共5页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金面上项目(81170466);国家重点基础研究发展计划(973计划)资助项目(2015CB964902)

摘　　要：目的探讨拟胚体(EB)造血相关表面标志物的变化及其向红细胞定向诱导分化的潜能。方法利用悬浮培养体系使人胚胎干细胞(h ESCs)形成三维囊状结构(EB),通过添加细胞因子BMP-4、SCF和FL使EB向造血前体细胞分化,通过流式细胞术(FACs)检测细胞发育各个阶段内皮及血细胞表面分子的变化情况以判断细胞的不同发育阶段。采用胰酶消化处理的方式将EB消化为单个细胞并向红细胞诱导分化,在诱导的d7、d14、d21取样,流式检测分析红细胞的成熟程度。结果在EB培养体系中,d6即可检测到造血前体细胞表面标志物CD34和CD31,且在EB培养的d6-d21,CD34+CD31+细胞的比例逐渐升高,到d15达到顶峰(8.86%)。在红细胞定向分化过程中,红细胞的纯度达73.22%,免疫荧光染色显示:ε亚基的阳性率为88%(424/482),γ亚基的阳性率为94%(326/347),而β亚基的阳性率为0%(0/167)。结论建立了1种EB悬浮培养体系和体外诱导红细胞的方法;EB的造血相关表面标志物的表达呈现时序性变化,且由EB分化而来的红细胞具有早期胚胎红细胞的发育特点。Objective To investigate the transition of hematopoietic surface marker of embryoid body （EB） and erythroid differentiation potential of EB. Methods Suspension culture system of human embryonic stem cells was applied to form three-dimensional cystic structure （EB）. BMP-4, SCF and FL were supplemented to induce EB to differentiate into hematopoiefic progenitor cells. The continuous changes of the cndothelium and hematopoiesis surface marker were verified by flow cytometry. Then, the development phases of the cells were classified. Cells were digested into single cells by trypsin and induced to erythrocytes. The extent of differentiation of erythrecytes was determined by sampling the cells at day 7, 14 and 21, followed by an analysis by flow cytometry. Result On day 6 of EB suspension culture, cells had already expressed hematopoietic precursor surface markers CD34 and CD31, which elevated from d6 to d21, and reached peak on day 15 （8. 86% ）. Among the cultured erythrocytes, the purity of the erythrocytes can be as high as 73.22%. The positive percentage of 8 and γ hemoglobin subsets were 88% （424/482） and 94% （326/347）, whereas 13 hemoglobin was 0% （0/167）. Conclusion A EB culturing system and a novel method were established in vitro to induce the differentiation into erythrocytes. The hemafopoietic surface marker manifest in a time-dependent manner during EB differentiation, and embryoid body derived erythrocytes display early fetal red blood cell characteristics.

关 键 词：人胚胎干细胞(hESCs) 拟胚体(EB) 造血表面标志 红细胞 悬浮培养 体外诱导 

分 类 号：R457.7[医药卫生—治疗学] R331[医药卫生—临床医学]