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

发布时间：2018-03-26 07:35

本文选题：人胚胎干细胞(hESCs)　切入点：拟胚体(EB)　出处：《中国输血杂志》2015年05期

【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。
[Abstract]:Objective to investigate the changes of hematopoietic associated surface markers and their potential to induce differentiation into erythrocytes. Methods suspension culture system was used to induce human embryonic stem cells (ESC) to form a three-dimensional cystic structure. Factor BMP-4 SCF and FL induced EB to differentiate into hematopoietic precursor cells. Flow cytometry (FACs) was used to detect the changes of endothelial cells and blood cell surface molecules in different stages of cell development. EB was digested into a single cell and differentiated into erythrocytes by trypsin digestion. The mature degree of erythrocytes was analyzed by flow cytometry. Results in EB culture system, the surface markers of hematopoietic precursor cells (CD34 and CD31) could be detected on the 6th day, and the proportion of CD34 CD31 cells in EB cultured on d6-d21 + CD34 was gradually increased. By the end of the day 15, the peak was 8.86. In the process of erythrocyte directional differentiation, The purity of erythrocyte was 73.22. The positive rate of 蔚 subunit was 884 / 482%, the positive rate of 纬 subunit was 9420 / 347%, and the positive rate of 尾 subunit was 0% / 167 ~ 0.Conclusion A EB suspension culture system and a method for inducing erythrocyte in vitro were established. The expression of hematopoietic related surface markers showed a temporal change. The erythrocytes differentiated from EB have the characteristics of early embryonic erythrocyte development.
【作者单位】：中国医学科学院北京协和医学院输血研究所;四川新生命干细胞科技股份有限公司;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;
【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)
【分类号】：R329

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