本文選題：人胚胎干細(xì)胞(hESCs)　切入點：擬胚體(EB)　出處：《中國輸血雜志》2015年05期

【摘要】：目的探討擬胚體(EB)造血相關(guān)表面標(biāo)志物的變化及其向紅細(xì)胞定向誘導(dǎo)分化的潛能。方法利用懸浮培養(yǎng)體系使人胚胎干細(xì)胞(h ESCs)形成三維囊狀結(jié)構(gòu)(EB),通過添加細(xì)胞因子BMP-4、SCF和FL使EB向造血前體細(xì)胞分化,通過流式細(xì)胞術(shù)(FACs)檢測細(xì)胞發(fā)育各個階段內(nèi)皮及血細(xì)胞表面分子的變化情況以判斷細(xì)胞的不同發(fā)育階段。采用胰酶消化處理的方式將EB消化為單個細(xì)胞并向紅細(xì)胞誘導(dǎo)分化,在誘導(dǎo)的d7、d14、d21取樣,流式檢測分析紅細(xì)胞的成熟程度。結(jié)果在EB培養(yǎng)體系中,d6即可檢測到造血前體細(xì)胞表面標(biāo)志物CD34和CD31,且在EB培養(yǎng)的d6-d21,CD34+CD31+細(xì)胞的比例逐漸升高,到d15達(dá)到頂峰(8.86%)。在紅細(xì)胞定向分化過程中,紅細(xì)胞的純度達(dá)73.22%,免疫熒光染色顯示:ε亞基的陽性率為88%(424/482),γ亞基的陽性率為94%(326/347),而β亞基的陽性率為0%(0/167)。結(jié)論建立了1種EB懸浮培養(yǎng)體系和體外誘導(dǎo)紅細(xì)胞的方法;EB的造血相關(guān)表面標(biāo)志物的表達(dá)呈現(xiàn)時序性變化,且由EB分化而來的紅細(xì)胞具有早期胚胎紅細(xì)胞的發(fā)育特點。
[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 緯 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.
【作者單位】： 中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院輸血研究所;四川新生命干細(xì)胞科技股份有限公司;中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院血液學(xué)研究所實驗血液學(xué)國家重點實驗室;
【基金】：國家自然科學(xué)基金面上項目(81170466) 國家重點基礎(chǔ)研究發(fā)展計劃(973計劃)資助項目(2015CB964902)
【分類號】：R329

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本文編號：1666988