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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 changes of hematopoietic related surface markers of embryoid body (EB) and its potential for directional differentiation into erythrocytes. Methods Human embryonic stem cells (hESCs) were induced into three-dimensional cystic structures (EBs) by suspension culture. EBs were differentiated into hematopoietic progenitor cells by addition of cytokines BMP-4, SCF and FL. Flow cytometry (FACs) Detection of cell growth in all stages of endothelial and blood cell surface molecules to determine the different stages of cell development. EBs were digested by trypsinization into single cells and differentiated into erythrocytes. Samples were taken on d7, d14 and d21, and the maturation of erythrocytes was analyzed by flow cytometry. Results In EB culture system, hematopoietic progenitor cell surface markers CD34 and CD31 were detected in d6, and the proportion of d6-d21 and CD34 + CD31 + cells in EB increased gradually to d15 (8.86%) . The purity of erythrocytes was 73.22% during erythroid differentiation. The positive rate of ε subunit was 88% (424/482) and the positive rate of γ subunit was 94% (326/347) by immunofluorescence staining. The positive rate of β subunit was 0% (0/167). Conclusion One kind of EB suspension culture system and the method of inducing erythrocytes in vitro were established. The expression of hematopoietic related surface markers of EB presented a time-series change, and erythrocytes differentiated from EB had the characteristics of development of early embryonic erythrocytes.