基于材料表面图案化技术研究细胞形状和表面手性特征对干细胞黏附与分化的影响

发布时间：2018-03-30 19:11

本文选题：高分子水凝胶　切入点：生物医用材料　出处：《复旦大学》2014年博士论文

【摘要】：新一代生物材料的设计在很大程度上依赖人类对细胞-材料相互作用规律的认识。如果将细胞外基质(ECM)和邻近的其它细胞都看作是广义“材料”的话,我们可以认为细胞本身就生存于“材料”世界里。生物体内的细胞无时无刻不与“材料”发生接触和相互作用,且细胞特定功能的实现在很大程度上也依赖于其所处的细胞微环境。生物材料学中的一个核心任务便是对细胞微环境进行仿生,即通过外界材料的设计与加工来模拟体内ECM的结构与功能,从而帮助组织或器官获得快速而有效的修复。因而,研究并揭示细胞-材料相互作用中各种因素的作用效应和机理在生物材料学、细胞生物学、组织工程和再生医学中都极为重要,也是生物医用材料中共性的重大科学问题。在传统的细胞培养体系中,影响细胞-材料相互作用的多种因素(细胞因素、细胞外基质因素和可溶性因子)往往混杂在一起而难以分开。得益于先进材料的发展,材料表面图案化技术可用于构建具有细胞黏附反差特性的图案,进而达到对细胞黏附的精确控制。该技术有望用于单独研究各种因素对细胞黏附、迁移、增殖和分化等行为的影响,从而为深入准确地理解细胞-材料相互作用提供独到的手段。骨髓基质干细胞具有自我复制更新的能力和多向诱导分化潜能,且可以取自于成体而不涉及伦理问题,因而成为再生医学和组织工程中理想的种子细胞类型。在干细胞相关的研究领域,考察并了解调节干细胞分化的各种因素一直是科学家关注的焦点,这些因素的组合应用能够最大程度地保障干细胞朝着需要的方向进行定向分化。本博士论文以骨髓基质干细胞为模型细胞,借助于独到的聚乙二醇(PEG)高分子水凝胶图案化技术和分子自组装技术研究了细胞形状、基底表面的分子手性特征(分子旋光性)和几何手性特征对干细胞黏附与分化行为的影响。本文的主要创新性工作和科学发现包含以下几个方面：(1)发展了聚乙二醇水凝胶表面制备细胞黏附微阵列的技术,获得了具有强烈细胞黏附反差的图案化表面,实现了对细胞形状的持久控制,并首次揭示细胞形状是调控干细胞分化的一种内在因素。发展了图案转移技术,设计并制备了具有持久抗细胞黏附反差的PEG水凝胶微米图案,为单细胞形状的持久控制提供了强有力的材料手段。利用该图案化技术,本研究在国际上首次实现了对细胞形状长达19天的控制。发现干细胞形状在诱导液和基础培养液环境下均能显著影响其分化行为：成骨分化在长径比为2左右达到峰值,而成脂分化随着长径比的增加单调下降。此外,本文还对这一形状效应发挥作用的调控机理作了探讨,首次揭示并确证了形状效应是一种内在的、独立于可溶性诱导因子的影响细胞分化行为的因素。这也表明在不借助于外界任何可溶诱导因子的条件下,可以通过单独的材料手段实现对干细胞形状的控制进而调控其分化行为。(2)综合有无图案化分子手性表面的制备,首次考察了材料表面分子的旋光性对干细胞行为的影响,发现分子手性特征可以显著影响干细胞的黏附和分化。利用L型半胱氨酸和D型半胱氨酸在金表面的接枝修饰和独特抗细胞黏附反差水凝胶微米图案的制备,本研究同时设计并制备了分子手性(L型和D型)表面与分子手性物质的微图案,并在这些表面对骨髓基质干细胞进行了培养与诱导分化。研究结果首次揭示了材料的分子手性特征能显著影响干细胞的黏附与分化行为。更深入的研究表明,材料表面的分子手性特征很可能是由于首先导致了蛋白吸附及细胞黏附面积的差异(对应于不同的细胞应力),进而影响了干细胞的分化行为。(3)设计并制备了具有几何手性微米岛的PEG水凝胶表面,首次探讨了细胞黏附微环境的几何手性特征对干细胞行为的影响,并发现该几何手性特征是细胞极性和分化的调控因素。在持久抗细胞黏附的PEG水凝胶表面成功设计、并制备了具有几何手性特征的螺线图案(顺、逆时针旋转)。根据对几何手性图案表面单细胞黏附和分化状况的统计,本研究首次发现,基底螺线图案的手性特征(顺、逆时针旋转)能够显著影响干细胞的极性取向和分化行为。进一步分析表明,细胞在几何手性微米图案上经“诱导”产生的细胞极性和细胞骨架可能存在的“固有手性特征”共同作用导致了顺时针和逆时针图案上细胞应力的差异,进而最终影响了干细胞的分化行为。顺时针图案上干细胞应力相对较大而利于成骨分化,而逆时针图案上干细胞应力相对较小而利于成脂分化。本文借助独到的高分子材料技术探讨了原本相互交织、难以阐明的细胞-材料相互作用中的若干基本科学问题。相关研究尤其拓展了对于干细胞行为调控因素的认识,这为深入理解细胞-材料相互作用这个经典难题提供了新的思路与方法,也为新型生物医用材料的设计和研发奠定了部分理论基础。
[Abstract]:The design of a new generation of biological materials depends on our understanding of the cell material interaction law to a great extent. If the extracellular matrix (ECM) and adjacent other cells are considered as generalized "material", we can conclude that the cell itself to survive in the material world. In vivo cell every hour and moment does not contact and interaction with the "material", and the cell specific functions are also largely dependent on the cellular microenvironment. A core task in biomaterial is the cellular microenvironment of bionics, through the design and processing of external materials to simulate the structure and function of in ECM, so as to help the tissues or organs of fast and effective repair. Therefore, all kinds of research and reveals the cell material interaction factors in the effect and mechanism in biomaterials, cell growth Material science, tissue engineering and regenerative medicine are extremely important, but also important scientific problems of common biomedical materials. In the traditional cell culture system, the influence of various factors of cell material interaction (cell factors, extracellular matrix and soluble factors) are often mixed and difficult to separate together. Thanks in advanced materials, material surface patterning technique can be used to construct cell adhesion contrast characteristic pattern, and achieve precise control of cell adhesion. The technology is expected to be used to separate the effects of various factors on cell adhesion, migration, proliferation and differentiation effects of behavior, so as to further accurate understanding of cell material interactions provide original means. Bone marrow stromal stem cells with self replication ability to self-renewal and multi-directional differentiation potential, and can be taken from the body and do not involve ethical issues, because A seed cell type of regenerative medicine and tissue engineering. The ideal in the research field of stem cells, investigate and understand the factors that regulate stem cell differentiation has been the focus of attention of the scientists, using a combination of these factors to maximize the protection of stem cell differentiation towards the direction you need. In this doctoral dissertation bone marrow stromal stem cells as a model cell, with the help of the original polyethylene glycol (PEG) hydrogel patterning techniques and molecular self-assembly technology of cell shape, molecular characteristics of chiral substrates (molecular rotation) and geometric features of the influence of chiral dry adhesion and differentiation of cell behavior. In this paper, the main innovative work and scientific discoveries include the following aspects: (1) the development of the hydrogel surface preparation of cell adhesion microarray technology, obtained with a strong cell The patterned surface adhesion contrast, to achieve a lasting control of cell shape, and for the first time to reveal the cell shape is a kind of intrinsic factors of stem cell differentiation regulation. The development of pattern transfer technology, designed and fabricated with durable resistance to cell adhesion contrast PEG hydrogel micron pattern, provides a powerful means for sustainable control of materials single cell shape. Using the patterning technology, this study for the first time in the world to achieve a control of 19 days on the cell shape. Stem cells in shape and induced liquid basal medium environment can significantly affect the differentiation of osteogenic differentiation: in length to diameter ratio is about 2 peak and fat with the increase of the differentiation ratio of length to diameter decreases monotonically. In addition, this paper also on the shape effect play a regulatory mechanism is discussed, and the effect was revealed for the first time the shape is an intrinsic, independent Factors affecting cell differentiation in soluble inducer. It also shows that in without the help of any external factors induced by soluble condition, can separate material means to control the shape of stem cells and regulating the differentiation behavior. (2) there is no comprehensive patterned surface preparation of chiral molecules, was studied for the first time the effect of material surface molecular rotation of stem cell behavior, molecular chirality can significantly affect cell adhesion and differentiation of stem. In grafted and modified gold surface and unique anti cell adhesion contrast hydrogel micro patterns were prepared by the L type D cysteine and homocysteine, this research and design and preparation of chiral molecules (type L and type D) micro patterned surface and molecular chiral substances, and in the surface of bone marrow stromal cells were cultured and induced to differentiate. The results revealed for the first time The molecular characteristics of chiral materials can significantly affect the adhesion and differentiation of stem cells. Further studies showed that the molecular characteristics of chiral material surface is likely to be due to the first lead to differences in the protein adsorption and cell adhesion area (corresponding to different cell stress), further affect the differentiation of stem cells (behavior. 3) design and PEG hydrogel surface geometry is prepared by chiral micron Island, for the first time to investigate the effect of geometric characteristics of chiral cell adhesion microenvironment of stem cell behavior, and found that the geometrical features are chiral regulation factors of polarity and differentiation of cells. In PEG hydrogel surface design and durable resistance to cell adhesion. The preparation of the spiral pattern has a geometric chiral character (CIS, counterclockwise). According to the statistics of the geometric pattern on the surface of single chiral cell adhesion and differentiation status, first discovered in this research, the basal spiral graph In the case of chiral features (CIS, counterclockwise) can significantly affect the polarity orientation and differentiation of stem cells. Further analysis showed that the cells in the geometric pattern on the chiral micron "induced" generation of cell polarity and cytoskeleton may exist in the "inherent chirality" jointly caused the clockwise and counter clockwise the pattern on the cell stress difference, and ultimately affect the differentiation of stem cell behavior. Clockwise pattern on the stem cell stress is relatively large and conducive to osteogenic differentiation, and counterclockwise pattern on the stem cell stress is relatively small and conducive to adipogenic differentiation. With the unique technology of polymer materials originally intertwined. Some basic problems difficult to elucidate the cell material interactions in related research. Especially expand understanding of stem cell behavior regulating factors, the in-depth understanding of cell material interaction This classic problem provides new ideas and methods, also laid the theoretical foundation for the research and design of new biomedical materials.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08;O631.11

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