本文選題：擬胚體 + 人胚胎干細(xì)胞　； 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2017年博士論文

【摘要】：目的:人干細(xì)胞因有強(qiáng)大的自我更新能力和多向分化潛能,利用小分子化合物調(diào)節(jié)效應(yīng)迅速可逆的特點(diǎn),從小分子化合物促進(jìn)胚胎干細(xì)胞存活、自我更新及分化發(fā)育等方面入手,深入了解其對(duì)胚胎干細(xì)胞的誘導(dǎo)作用,以此探討在體外將胚胎干細(xì)胞誘導(dǎo)分化為角膜上皮樣細(xì)胞的方法,進(jìn)一步揭示在誘導(dǎo)過(guò)程中相應(yīng)的分子機(jī)制。本課題設(shè)計(jì)了兩組小分子化合物組合作為不同的誘導(dǎo)方式,通過(guò)不同的檢測(cè)方法證實(shí)誘導(dǎo)生成的細(xì)胞為角膜上皮樣細(xì)胞,為角膜細(xì)胞移植治療獲得無(wú)限制角膜上皮來(lái)源,減少臨床對(duì)受限于角膜供體的角膜移植手術(shù)需求,使更多患者獲得有效和及時(shí)治療提供參考途徑。方法:人胚胎干細(xì)胞在體外培養(yǎng)至融合度到達(dá)80%以上消化,使用Aggrewell孔板培養(yǎng)法獲得擬胚體(Embryonic bodies, EBs),在不同的誘導(dǎo)培養(yǎng)基的作用下加入小分子化合物誘導(dǎo)在體外可以分化為各種類型細(xì)胞的擬胚體向外胚層、角膜上皮祖細(xì)胞及角膜上皮細(xì)胞方向分化。擬胚體在低粘附六孔板中誘導(dǎo)培養(yǎng)4天后貼壁培養(yǎng)。隨著誘導(dǎo)時(shí)間增加,觀察從擬胚體爬出的細(xì)胞的形態(tài),留取誘導(dǎo)第10天、20天、30天的細(xì)胞行細(xì)胞免疫熒光檢測(cè),提取RNA和蛋白質(zhì),從DNA和蛋白水平檢測(cè)胚胎干細(xì)胞多能性基因OCT4、PAX6;角膜上皮祖細(xì)胞表面標(biāo)記物K15和角膜上皮細(xì)胞標(biāo)記物K12、K3表達(dá)水平。探索不同小分子和不同誘導(dǎo)培養(yǎng)基組合誘導(dǎo)特點(diǎn)及分化效率,尋求最優(yōu)誘導(dǎo)效率方式。結(jié)果:1.小分子化合物IWP-2、SB505124和DAPT不同組合可在體外誘導(dǎo)人胚胎干細(xì)胞分化為角膜上皮樣細(xì)胞;2.隨著誘導(dǎo)時(shí)間增加,小分子化合物誘導(dǎo)組細(xì)胞胚胎干細(xì)胞多能性基因的表達(dá)逐漸下調(diào)而角膜上皮祖細(xì)胞和角膜上皮細(xì)胞表面標(biāo)記物的表達(dá)逐漸上調(diào);3.SHEM培養(yǎng)基誘導(dǎo)分化作用下IWP-2、SB505124組合在第20天角膜上皮標(biāo)記物K3、K12的表達(dá)量最大。UM培養(yǎng)基誘導(dǎo)分化作用下IWP-2、SB505124組合和IWP-2、SB505124、DAPT組合在第20天誘導(dǎo)分化效率最高。DKSFM培養(yǎng)基誘導(dǎo)分化作用下IWP-2、SB505124、DAPT組合在第30天誘導(dǎo)分化效率最高。結(jié)論:1.小分子化合物誘導(dǎo)刺激后從擬胚體爬出的細(xì)胞具有角膜上皮細(xì)胞形態(tài)學(xué)的特點(diǎn),并且隨著誘導(dǎo)時(shí)間增加細(xì)胞的數(shù)量逐漸增加;2.免疫熒光、PCR和WB結(jié)果提示隨著誘導(dǎo)時(shí)間增加,小分子化合物誘導(dǎo)組細(xì)胞胚胎干細(xì)胞多能性基因的表達(dá)消失而角膜上皮祖細(xì)胞和角膜上皮細(xì)胞表面標(biāo)記物的表達(dá)出現(xiàn);3.不同的小分子化合物組合在不同誘導(dǎo)培養(yǎng)基的作用下的誘導(dǎo)效率不同;4.相同誘導(dǎo)培養(yǎng)基不相同小分子化合物在不同的誘導(dǎo)階段誘導(dǎo)效率存在差異。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R77

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相關(guān)期刊論文 前2條

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本文編號(hào)：1884904