微图案化表面的制备以及细胞间接触影响干细胞分化的研究

发布时间：2018-05-27 10:04

本文选题：再生医学 + 干细胞分化　；参考：《复旦大学》2010年博士论文

【摘要】： 调控细胞行为、尤其细胞黏附和干细胞分化是再生医学领域的一个研究重点,涉及到细胞生物学、医学、材料学等学科知识。迄今已有许多研究考察了生物材料模拟细胞外基质的物理、化学等特性去控制细胞的黏附,进而调控细胞的生命活动。但对细胞与细胞之间接触如何影响干细胞分化的研究则相对很少,已有研究的结论也有待确证,且不能定量给出接触程度与分化程度之间的关系。这并非因为细胞间接触对细胞生命活动不重要,在很大程度上是由于缺乏必要的研究技术手段,很难排除细胞密度变化导致的细胞自身分泌的因子浓度变化等因素的干扰。传统的细胞研究方式,比如在培养板上研究细胞,难以克服细胞迁移等问题,新发展起来的微图案技术则可以实现细胞定位,有助于研究细胞间接触等影响细胞生命活动的相关问题。本论文尝试了多种微图案制备方法,最终从中寻找到了一种能长时间阻止细胞迁移的微图案。我们以此首次定量考察了细胞接触程度对于干细胞定向分化的影响。我们的这些实验技术和方法,以及细胞研究结论不仅丰富了细胞学知识,也为细胞学研究提供了新的手段和途径；对再生医学领域里面诸如组织修复材料的设计、干细胞调控等提供了一些基本的实验和理论知识。本论文的主要创新性研究包括以下几方面： 1.尝试了多种微图案制备方法,包括在PLGA膜上制备微拓扑形貌、PEG接枝表面制备有细胞黏附反差的化学微图案、PEG水凝胶形成的微凹坑图案、PEG水凝胶表面的化学微图案等。每种方法都反复摸索了详细的制备条件,根据细胞定位实验,从中确定了PEG水凝胶表面的化学微图案能够长时间控制细胞黏附位置,特别是阻止了细胞的迁移等运动,为后续的干细胞研究奠定了基础。其它几种方法制备的微图案,尽管不能长时间限制细胞的黏附位置,但也有各自的特点,适合于相应的细胞研究,例如微拓扑形貌可以调节细胞铺展；PEG接枝的表面可以考察短时间内的细胞形态、黏附、迁移等问题。 2.建立了利用光学显微镜照片分析细胞内碱性磷酸酶活性的半定量分析方法。骨髓基质干细胞被定位在微图案上后成骨分化,但在同一微图案上面存在不同的细胞接触状况,比如有的细胞处于孤立状态；有的接触了一个细胞；有的接触了两个,乃至多个不等的细胞,它们的成骨分化程度存在差异,却又难以用直接测吸光度的方法来判别。比照分光光度法,我们通过染色、拍照,测量细胞灰度值,并根据吸光值计算公式,定量分析了不同细胞接触情况下的干细胞成骨分化水平。虽然我们是针对骨髓基质干细胞的成骨分化所建立的分析方法,但这个方法同样适用于其它的,能够通过染色来定性、定量分析的研究中。 3.利用之前确定的微图案制备技术以及建立的半定量分析方法,我们考察了细胞接触对大鼠骨髓基质干细胞成骨分化和成脂分化的影响。我们在同一基底材料表面利用不同黏附圆岛导致不同细胞接触程度的技术排除了细胞密度变化导致因子浓度不同等因素的干扰。我们的结论确证了细胞接触能够促进干细胞定向分化,并首次发现这种促进作用与细胞接触程度成正比。我们还发现缝隙连接是相关细胞接触的重要因素,但并非唯一因素。
[Abstract]:Regulation of cell behavior, especially cell adhesion and stem cell differentiation, is a research focus in the field of regenerative medicine. It involves the knowledge of cell biology, medicine and material science. So far, many studies have studied the physical, chemical and chemical properties of biomaterials to control cell adhesion to control cell life. But there are few studies on how cell and cell contact affect stem cell differentiation. The conclusion of the study has yet to be confirmed, and the relationship between the degree of contact and the degree of differentiation can not be quantified. This is not due to the lack of intercellular contact to cell life activity, to a large extent, due to the lack of necessary research. By means of technology, it is difficult to exclude the interference of factors such as the concentration of cytokines secreted by cell density changes.
The traditional method of cell research, such as studying cells on the culture plate, is difficult to overcome cell migration. The newly developed micro pattern technology can realize cell location and help to study the intercellular contact and other related problems that affect cell life activity. A micro pattern that prevents cell migration for a long time. For the first time, we examine the effect of cell contact on the directional differentiation of stem cells. Our experimental techniques and methods, as well as the conclusions of cell research not only enrich the knowledge of cytology, but also provide new means and ways for the study of cytology. Some basic experimental and theoretical knowledge is provided in the field of learning such as tissue repair materials design, stem cell regulation and so on.
The main innovative research in this paper includes the following aspects:
1. a variety of micro pattern preparation methods, including the preparation of microtopologies on the PLGA membrane, the preparation of chemical micropatterns with cell adhesion contrast, the micropits pattern formed by the PEG hydrogel and the chemical micropatterns on the surface of the PEG hydrogel, were prepared on the PEG graft surface. It is determined that the chemical micropatterns on the surface of the PEG hydrogel can control the cell adhesion for a long time, especially to prevent the migration of cells, which lays the foundation for the subsequent research of stem cells. The micropatterns made by other methods, although not long time limit the adhesion of cells, have their own characteristics, suitable for the phase. Cell research, such as micro topology, can regulate cell spreading. The surface of PEG grafted can be used to investigate cell morphology, adhesion and migration in a short time.
2. a semi quantitative analysis method was established to analyze the activity of alkaline phosphatase in cells by a photo microscope. The bone marrow stromal cells were differentiated into bone after being located on the micropatterns, but there were different cell contact conditions on the same micro pattern, such as some cells were in isolation; some were exposed to a cell; some were connected. Two, and even many different cells, the differentiation degree of their osteogenesis is different, but it is difficult to discriminate by direct measurement of absorbency. Although we are an analytical method for osteogenic differentiation of bone marrow stromal cells, this method is also applicable to other studies that can be qualitatively and quantitatively analyzed by dyeing.
3. the effects of cell contact on osteogenesis and lipid differentiation of bone marrow stromal cells in rats were investigated by using previously determined micro pattern preparation techniques and semi quantitative analysis methods. Our conclusions confirm that cell contact can promote stem cell differentiation, and that this promotion is in proportion to the degree of cell contact for the first time. We also found that gap junction is an important factor in cell contact, but it is not the only factor.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【相似文献】