人羊膜来源干细胞的性质及分化潜能研究

发布时间：2018-06-24 20:43

本文选题：人羊膜来源干细胞 + 多能干细胞　；参考：《中国协和医科大学》2008年博士论文

【摘要】： 背景基于干细胞研究的再生医学有望修复心肌损伤,是当前研究的热点。人羊膜来源干细胞具有多向分化潜能,且因其易于获得、不引起伦理学争议、干细胞含量丰富、免疫原性低等特点,有潜力成为未来再生医学临床应用的重要种子细胞来源。目的 1.了解人羊膜不同部位(层面)来源干细胞的性质差异。 2.评价RCCS(旋转细胞培养系统)等培养方式在人羊膜来源干细胞培养中的价值。 3.比较不同方法诱导人羊膜来源干细胞向心肌细胞方向分化的效果。方法与结果 1.羊膜上皮层来源的细胞表达各种干细胞标记物的水平普遍高于羊膜间质层来源的间充质干细胞;前者仅CD73表达水平低于后者。而羊膜上皮层内,近基底部的上皮细胞表达干细胞标记物的水平均高于近表层部(流式细胞学分析方法)。 2.RCCS培养较静止培养更利于维持各种干细胞标记物的表达水平(流式细胞学分析方法)。RCCS培养羊膜上皮细胞2周后,细胞倾向聚集生长,并有类胚体样细胞团形成,其中存在高表达胚胎干细胞标记物的细胞(免疫组化法、免疫荧光法)。 3.以心肌细胞裂解液、GDF-15、BMP2+FGF10分别对羊膜来源干细胞进行诱导,2周后各诱导组在蛋白水平及mRNA水平不同程度的表达心肌标记物(免疫组化法,RT-PCR法)。GDF-15诱导组表达MEF2C及GATA-4水平较高,BMP2+FGF10组表达HCN2水平较高,仅心肌细胞裂解液组明确表达KIR2.1。 4.羊膜来源干细胞具有不对称分裂的特点,以体积较大、形态较圆、胞内颗粒样物质较丰富明显的细胞表达干细胞标记物水平较高;而体积偏小、形态近圆或略不规则的细胞表达功能蛋白较强(免疫组化法、免疫荧光法)。结论 1.羊膜来源干细胞的性质及分布存在不均一性,这为进一步筛选更具临床应用价值的细胞来源提供了依据。 2.RCCS培养有助于维持人羊膜来源干细胞的多向分化潜能。 3.诱导人羊膜来源干细胞向心肌细胞方向分化的过程中,GDF-15具有一定作用。干细胞向心肌细胞方向分化可能需要多种细胞因子的共同作用。
[Abstract]:Background Regenerative medicine based on stem cell research is a hot topic in recent years, which is expected to repair myocardial injury. Human amniotic membrane derived stem cells (HMSCs) have the potential to become an important source of seed cells for the clinical application of regenerative medicine because they are easy to obtain and do not cause ethical controversy, the content of stem cells is rich, and the immunogenicity is low. Objective 1. To understand the nature differences of stem cells derived from different parts of human amniotic membrane. 2. To evaluate the value of RCCS (rotating cell culture system) in the culture of human amniotic stem cells. To compare the effects of different methods on inducing human amniotic stem cells to differentiate into cardiomyocytes. Methods and results 1. The expression of various stem cell markers in the cells derived from the amniotic epithelium was generally higher than that in the mesenchymal stem cells derived from the amniotic mesenchymal layer, and the expression level of CD73 in the former was lower than that in the latter. In the epithelial layer of amniotic membrane, The expression levels of stem cell markers in epithelial cells near the base were higher than those in the proximal surface (flow cytometric analysis) 2.RCCS culture was more conducive to maintaining the expression of stem cell markers than static culture. RCCS culture of amniotic epithelial cells for 2 weeks, Cells tend to gather and grow, and there are embryoid cell clusters, in which there are cells that highly express the markers of embryonic stem cells (immunohistochemical method, immunofluorescence method). 3. Expression of Myocardial Marker at different levels of protein and mRNA in different groups (Immunohistochemical RT-PCR). GDF-15 induced expression of MEF2C and GATA-4 water in amniotic stem cells induced by GDF-15 BMP2FGF10 for 2 weeks The level of HCN2 expression was higher in BMP2FGF10 group. Only myocardial cell lysate group expressed KIR 2.1.4. Amniotic membrane derived stem cells have the characteristics of asymmetric division. The cells with larger size, more round shape, more abundant granular substances in the cell have higher expression level of stem cell markers, but the volume of stem cells is relatively small, while amniotic membrane derived stem cells have the characteristics of asymmetric division. Cells with near-round or slightly irregular morphology expressed strong functional proteins (immunohistochemical method, immunofluorescence method). Conclusion 1. The nature and distribution of amniotic stem cells were not uniform. 2. RCCS culture is helpful to maintain the multidirectional differentiation potential of human amniotic stem cells. 2. GDF-15 plays an important role in inducing human amniotic stem cells to differentiate into cardiomyocytes. Differentiation of stem cells into cardiomyocytes may require multiple cytokines.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

【共引文献】