本文選題：高分子水凝膠　切入點：生物醫(yī)用材料　出處：《復旦大學》2014年博士論文

【摘要】：新一代生物材料的設計在很大程度上依賴人類對細胞-材料相互作用規(guī)律的認識。如果將細胞外基質(ECM)和鄰近的其它細胞都看作是廣義“材料”的話,我們可以認為細胞本身就生存于“材料”世界里。生物體內的細胞無時無刻不與“材料”發(fā)生接觸和相互作用,且細胞特定功能的實現在很大程度上也依賴于其所處的細胞微環(huán)境。生物材料學中的一個核心任務便是對細胞微環(huán)境進行仿生,即通過外界材料的設計與加工來模擬體內ECM的結構與功能,從而幫助組織或器官獲得快速而有效的修復。因而,研究并揭示細胞-材料相互作用中各種因素的作用效應和機理在生物材料學、細胞生物學、組織工程和再生醫(yī)學中都極為重要,也是生物醫(yī)用材料中共性的重大科學問題。在傳統(tǒng)的細胞培養(yǎng)體系中,影響細胞-材料相互作用的多種因素(細胞因素、細胞外基質因素和可溶性因子)往往混雜在一起而難以分開。得益于先進材料的發(fā)展,材料表面圖案化技術可用于構建具有細胞黏附反差特性的圖案,進而達到對細胞黏附的精確控制。該技術有望用于單獨研究各種因素對細胞黏附、遷移、增殖和分化等行為的影響,從而為深入準確地理解細胞-材料相互作用提供獨到的手段。骨髓基質干細胞具有自我復制更新的能力和多向誘導分化潛能,且可以取自于成體而不涉及倫理問題,因而成為再生醫(yī)學和組織工程中理想的種子細胞類型。在干細胞相關的研究領域,考察并了解調節(jié)干細胞分化的各種因素一直是科學家關注的焦點,這些因素的組合應用能夠最大程度地保障干細胞朝著需要的方向進行定向分化。本博士論文以骨髓基質干細胞為模型細胞,借助于獨到的聚乙二醇(PEG)高分子水凝膠圖案化技術和分子自組裝技術研究了細胞形狀、基底表面的分子手性特征(分子旋光性)和幾何手性特征對干細胞黏附與分化行為的影響。本文的主要創(chuàng)新性工作和科學發(fā)現包含以下幾個方面：(1)發(fā)展了聚乙二醇水凝膠表面制備細胞黏附微陣列的技術,獲得了具有強烈細胞黏附反差的圖案化表面,實現了對細胞形狀的持久控制,并首次揭示細胞形狀是調控干細胞分化的一種內在因素。發(fā)展了圖案轉移技術,設計并制備了具有持久抗細胞黏附反差的PEG水凝膠微米圖案,為單細胞形狀的持久控制提供了強有力的材料手段。利用該圖案化技術,本研究在國際上首次實現了對細胞形狀長達19天的控制。發(fā)現干細胞形狀在誘導液和基礎培養(yǎng)液環(huán)境下均能顯著影響其分化行為：成骨分化在長徑比為2左右達到峰值,而成脂分化隨著長徑比的增加單調下降。此外,本文還對這一形狀效應發(fā)揮作用的調控機理作了探討,首次揭示并確證了形狀效應是一種內在的、獨立于可溶性誘導因子的影響細胞分化行為的因素。這也表明在不借助于外界任何可溶誘導因子的條件下,可以通過單獨的材料手段實現對干細胞形狀的控制進而調控其分化行為。(2)綜合有無圖案化分子手性表面的制備,首次考察了材料表面分子的旋光性對干細胞行為的影響,發(fā)現分子手性特征可以顯著影響干細胞的黏附和分化。利用L型半胱氨酸和D型半胱氨酸在金表面的接枝修飾和獨特抗細胞黏附反差水凝膠微米圖案的制備,本研究同時設計并制備了分子手性(L型和D型)表面與分子手性物質的微圖案,并在這些表面對骨髓基質干細胞進行了培養(yǎng)與誘導分化。研究結果首次揭示了材料的分子手性特征能顯著影響干細胞的黏附與分化行為。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導致了蛋白吸附及細胞黏附面積的差異(對應于不同的細胞應力),進而影響了干細胞的分化行為。(3)設計并制備了具有幾何手性微米島的PEG水凝膠表面,首次探討了細胞黏附微環(huán)境的幾何手性特征對干細胞行為的影響,并發(fā)現該幾何手性特征是細胞極性和分化的調控因素。在持久抗細胞黏附的PEG水凝膠表面成功設計、并制備了具有幾何手性特征的螺線圖案(順、逆時針旋轉)。根據對幾何手性圖案表面單細胞黏附和分化狀況的統(tǒng)計,本研究首次發(fā)現,基底螺線圖案的手性特征(順、逆時針旋轉)能夠顯著影響干細胞的極性取向和分化行為。進一步分析表明,細胞在幾何手性微米圖案上經“誘導”產生的細胞極性和細胞骨架可能存在的“固有手性特征”共同作用導致了順時針和逆時針圖案上細胞應力的差異,進而最終影響了干細胞的分化行為。順時針圖案上干細胞應力相對較大而利于成骨分化,而逆時針圖案上干細胞應力相對較小而利于成脂分化。本文借助獨到的高分子材料技術探討了原本相互交織、難以闡明的細胞-材料相互作用中的若干基本科學問題。相關研究尤其拓展了對于干細胞行為調控因素的認識,這為深入理解細胞-材料相互作用這個經典難題提供了新的思路與方法,也為新型生物醫(yī)用材料的設計和研發(fā)奠定了部分理論基礎。
[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.

【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R318.08;O631.11

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