诱导型多能干细胞在体外三维环境中诱导分化出肠道类器官

发布时间：2018-06-06 02:30

本文选题：干细胞 + 多能干细胞　；参考：《中国组织工程研究》2017年25期

【摘要】：背景:诱导型多能干细胞是一类特殊的细胞,具有自我更新及多向分化潜力,在一定诱导条件下能够分化为肠道类器官。目的:探究诱导型多能干细胞在体外特定条件下是否可分化出肠道类器官。方法:复苏B6J小鼠18代诱导型多能干细胞,培养3 d克隆单位覆盖培养皿80%左右后,加入含有激活素A的培养基培养3 d,得到确定性内胚层,加入含成纤维细胞生长因子4和Wnt3A的培养基培养4 d,得到中后肠细胞球,将其与Matrigel培养基混合后滴入四孔板形成滴株样生长,在HEPES、Rspondin1、头蛋白、表皮细胞生长因子、B27添加剂等生长因子等共同作用下向肠道类器官分化,期间观察细胞形态变化,检测确定性内胚层FoxA 2、Sox17表达,中后肠细胞球特异性标记物CDX2表达,肠道类器官特异性标记物Sox9、CGA、MMP7表达。结果与结论:(1)诱导型多能干细胞在含有激活素A的培养基培养3 d,细胞初步分化,相互融合,确定性内胚层标记物FoxA 2、Sox17表达较诱导型多能干细胞明显升高(P0.05);(2)确定性内胚层在含成纤维细胞生长因子4和WNT3A环境下培养第3天开始就有少量中后肠细胞球形成,第4天大量中后肠细胞球形成,中后肠细胞球标记物CDX2较确定性内胚层明显升高(P0.05);(3)中后肠细胞球培养3 d后逐渐形成肠道类器官,其特异性标记物Sox9、CGA、MMP7表达明显高于中后肠细胞球(P0.05);(4)结果表明,诱导型多能干细胞可在体外三维环境中诱导分化出类肠道器官。
[Abstract]:Background: inducible pluripotent stem cells, a special type of cells, have the potential of self renewal and multidirectional differentiation and can be differentiated into intestinal organs under certain induction conditions. Objective: To explore whether the inducible pluripotent stem cells can differentiate into intestinal organs under specific conditions in vitro. Methods: resuscitation of the 18 generation of inducible pluripotent stem cells in B6J mice, After the culture of 3 D clone unit covered culture dish 80%, the culture medium containing activin A was added to 3 D, the determinate endoderm was obtained, the medium culture medium containing fibroblast growth factor 4 and Wnt3A was added to 4 D, and the medium and posterior intestinal cell spheres were obtained, and then mixed with the Matrigel medium into the four orifice to form the drop strain growth, in HEPES, Rspondin. 1, head protein, epidermal growth factor, B27 additive and other growth factors, such as growth factors and other growth factors to differentiate into intestinal organs, during the period of observation of cell morphological changes, determination of the determinate endoderm FoxA 2, Sox17 expression, middle and posterior intestinal cell specific markers CDX2 expression, intestinal organ specific markers Sox9, CGA, MMP7 expression. (1: (1): (1) The inducible pluripotent stem cells were cultured in the medium containing activin A for 3 D, the cells were initially differentiated and fused, the determinate endoderm marker FoxA 2 and the expression of Sox17 were significantly higher than those of the inducible pluripotent stem cells (P0.05). (2) a small amount of the determinate endoderm began to occur in a small amount after third days of cultivation of fibroblast growth factor 4 and WNT3A environment. The formation of intestinal cell balls, a large number of midgut cell balls formed in fourth days, the medium and posterior gut cell marker CDX2 was significantly higher than that of the determinate endoderm (P0.05). (3) the intestinal cells were gradually formed after the culture of the middle and posterior intestinal cells of 3 D, and the specific markers, Sox9, CGA, and MMP7, were significantly higher than the middle and posterior intestinal cell spheres (P0.05); (4) the results showed induced pluripotent Stem cells can differentiate into intestinal like organs in three dimensional environment.
【作者单位】：南京大学医学院附属金陵医院解放军南京军区南京总医院解放军普通外科研究所;解放军第二军医大学;
【基金】：南京军区医药卫生科研基金(15DX018)~~
【分类号】：R329.2

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