小分子化合物诱导人胚胎干细胞分化为角膜上皮样细胞的研究

发布时间：2018-05-13 21:31

本文选题：拟胚体 + 人胚胎干细胞　；参考：《中国人民解放军医学院》2017年博士论文

【摘要】：目的:人干细胞因有强大的自我更新能力和多向分化潜能,利用小分子化合物调节效应迅速可逆的特点,从小分子化合物促进胚胎干细胞存活、自我更新及分化发育等方面入手,深入了解其对胚胎干细胞的诱导作用,以此探讨在体外将胚胎干细胞诱导分化为角膜上皮样细胞的方法,进一步揭示在诱导过程中相应的分子机制。本课题设计了两组小分子化合物组合作为不同的诱导方式,通过不同的检测方法证实诱导生成的细胞为角膜上皮样细胞,为角膜细胞移植治疗获得无限制角膜上皮来源,减少临床对受限于角膜供体的角膜移植手术需求,使更多患者获得有效和及时治疗提供参考途径。方法:人胚胎干细胞在体外培养至融合度到达80%以上消化,使用Aggrewell孔板培养法获得拟胚体(Embryonic bodies, EBs),在不同的诱导培养基的作用下加入小分子化合物诱导在体外可以分化为各种类型细胞的拟胚体向外胚层、角膜上皮祖细胞及角膜上皮细胞方向分化。拟胚体在低粘附六孔板中诱导培养4天后贴壁培养。随着诱导时间增加,观察从拟胚体爬出的细胞的形态,留取诱导第10天、20天、30天的细胞行细胞免疫荧光检测,提取RNA和蛋白质,从DNA和蛋白水平检测胚胎干细胞多能性基因OCT4、PAX6;角膜上皮祖细胞表面标记物K15和角膜上皮细胞标记物K12、K3表达水平。探索不同小分子和不同诱导培养基组合诱导特点及分化效率,寻求最优诱导效率方式。结果:1.小分子化合物IWP-2、SB505124和DAPT不同组合可在体外诱导人胚胎干细胞分化为角膜上皮样细胞;2.随着诱导时间增加,小分子化合物诱导组细胞胚胎干细胞多能性基因的表达逐渐下调而角膜上皮祖细胞和角膜上皮细胞表面标记物的表达逐渐上调;3.SHEM培养基诱导分化作用下IWP-2、SB505124组合在第20天角膜上皮标记物K3、K12的表达量最大。UM培养基诱导分化作用下IWP-2、SB505124组合和IWP-2、SB505124、DAPT组合在第20天诱导分化效率最高。DKSFM培养基诱导分化作用下IWP-2、SB505124、DAPT组合在第30天诱导分化效率最高。结论:1.小分子化合物诱导刺激后从拟胚体爬出的细胞具有角膜上皮细胞形态学的特点,并且随着诱导时间增加细胞的数量逐渐增加;2.免疫荧光、PCR和WB结果提示随着诱导时间增加,小分子化合物诱导组细胞胚胎干细胞多能性基因的表达消失而角膜上皮祖细胞和角膜上皮细胞表面标记物的表达出现;3.不同的小分子化合物组合在不同诱导培养基的作用下的诱导效率不同;4.相同诱导培养基不相同小分子化合物在不同的诱导阶段诱导效率存在差异。
[Abstract]:Objective: human stem cells have the ability of self-renewal and multidirectional differentiation, and promote the survival of embryonic stem cells from small molecular compounds by using the characteristics of small molecular compounds to regulate the effect quickly and reversible. In order to explore the methods of inducing embryonic stem cells into corneal epithelioid cells in vitro, the self-renewal and differentiation and development of embryonic stem cells were studied. The molecular mechanism in the induction process was further revealed. In this study, two groups of small molecular compounds were designed as different inductive methods. Different detection methods were used to confirm that the induced cells were corneal epithelioid cells, which provided an unlimited source of corneal epithelium for corneal cell transplantation. To reduce the clinical requirement of cornea transplantation limited by donor, so that more patients can get effective and timely treatment. Methods: human embryonic stem cells were cultured and digested to more than 80% in vitro. Embryonic bodieses (ESBs) were obtained by using Aggrewell pore plate culture method. The embryoid bodies of different types of cells could be induced to ectoderm by adding small molecular compounds in different induction media. Corneal epithelial progenitor cells and corneal epithelial cells differentiated in the direction. The embryoid was induced and cultured in a low adhesion six-hole plate for 4 days and then adhered to the wall. With the increase of induction time, the morphology of the cells crawling out of the embryoid body was observed, and the cells from the 10th day to the 20th day, 30 days after induction, were detected by immunofluorescence to extract RNA and protein. DNA and protein levels were used to detect the expression of multipotent gene OCT4PAX6, corneal epithelial progenitor cell surface marker K15 and corneal epithelial cell marker K12K3. To explore the characteristics and differentiation efficiency of different small molecules and different induction medium, and to find the best way of induction efficiency. The result is 1: 1. Different combinations of IWP-2SSB505124 and DAPT could induce the differentiation of human embryonic stem cells into corneal epithelioid cells in vitro. With the increase of induction time, The expression of pluripotent genes of embryonic stem cells induced by small molecular compounds was down-regulated and the expression of surface markers of corneal epithelial progenitor cells and corneal epithelial cells was gradually up-regulated. 3. The combination of IWP-2 and SB505124 was induced by SHEM medium. On the 20th day, the maximum expression of K3OK12 was found in the corneal epithelial marker. Under the induction of differentiation on the UM medium, the combination IWP-2SSB505124and IWP-2SSB505124DAPT had the highest induction and differentiation efficiency on the 20th day, and the combination of IWP-2SB505124DAPT had the highest differentiation efficiency on the 30th day under the inducement of differentiation on the medium of .DKSFM, and IWP-2SB505124DAPT was the best. Conclusion 1. The cells that crawled out of the embryoid body after stimulation by small molecular compounds had the morphological characteristics of corneal epithelial cells, and the number of cells increased gradually with the increase of induction time. The results of immunofluorescence PCR and WB indicated that the expression of pluripotent genes of embryonic stem cells disappeared and the expression of markers on the surface of corneal epithelial progenitor cells and corneal epithelial cells appeared with the increase of induction time. The induction efficiency of different combinations of small molecular compounds was different under different induction medium. The induction efficiency of different small molecular compounds in the same induction medium was different at different induction stages.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R77

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