發(fā)布時(shí)間：2018-11-08 08:51

【摘要】：細(xì)胞與材料相互作用是生物材料學(xué)、細(xì)胞生物學(xué)和再生醫(yī)學(xué)的一個(gè)基本科學(xué)問題,相關(guān)研究對(duì)于生物材料的設(shè)計(jì)具有重要的指導(dǎo)意義。細(xì)胞作為生命活動(dòng)的基本單元,對(duì)單細(xì)胞的研究能直接反映細(xì)胞內(nèi)本質(zhì)的變化。但由于高效的單細(xì)胞研究手段的相對(duì)不足,這方面的進(jìn)展仍然不夠。微米圖案技術(shù),能在基底上實(shí)現(xiàn)細(xì)胞黏附反差,從而能控制細(xì)胞的定位,經(jīng)調(diào)節(jié)細(xì)胞可黏附區(qū)域即黏附島的大小可以有效地實(shí)現(xiàn)單細(xì)胞的黏附,且樣本數(shù)量大,可用于統(tǒng)計(jì)而得到可信的結(jié)論,在對(duì)單細(xì)胞的研究中有很好的應(yīng)用前景。 組織器官不是由單細(xì)胞組成的,而是多個(gè)或多種細(xì)胞及胞外的基質(zhì)等組成的一個(gè)大社會(huì)。細(xì)胞與細(xì)胞之間相互作用也是組織工程中重要的基礎(chǔ)科學(xué)問題。多細(xì)胞的聚集程度,即細(xì)胞密度也非常重要。微米圖案技術(shù),通過控制細(xì)胞的定位,從而可以有效地控制細(xì)胞與細(xì)胞的接觸、細(xì)胞的聚集程度等,是研究多細(xì)胞之間相互作用的有效手段。 骨髓基質(zhì)干細(xì)胞具有多向分化潛能且易于體外擴(kuò)增,取自成體而不涉及倫理范疇,是組織工程理想的種子細(xì)胞類型。干細(xì)胞分化是當(dāng)今白然科學(xué)的研究前沿。 本博士論文選用骨髓基質(zhì)干細(xì)胞為模型,借助高分子基底上獨(dú)到的圖案化技術(shù)來研究影響干細(xì)胞分化的各種物理因素,包括單細(xì)胞分化的形狀效應(yīng)、尺寸效應(yīng),以及多細(xì)胞狀態(tài)下干細(xì)胞分化的密度效應(yīng)。 本論文的主要?jiǎng)?chuàng)新性工作包含以下幾方面： 1.制備了具有強(qiáng)烈細(xì)胞黏附反差的微圖案化表面的高分子水凝膠,并提出了適于細(xì)胞形狀研究的黏附島面積的判據(jù)以及基于顯微照片統(tǒng)計(jì)干細(xì)胞分化程度的半定量方法。論文工作過程中制備了聚乙二醇(PEG)水凝膠表面精氨酸—甘氨酸—天冬氨酸(RGD)微圖案,并借助于該高分子強(qiáng)烈抗拒細(xì)胞黏附的特征實(shí)現(xiàn)了細(xì)胞在黏附島上的良好而持久的定位。在此基礎(chǔ)上,建立了利用圖案化表面的細(xì)胞顯微照片判斷骨髓基質(zhì)干細(xì)胞成骨分化和成脂分化的統(tǒng)計(jì)方法,并且提出了最適于考察細(xì)胞形狀效應(yīng)的細(xì)胞可黏附的微島面積的判據(jù)。這樣,為成功實(shí)現(xiàn)不同形狀和長徑比的單細(xì)胞的定位,并合理、有效地基于微圖案化表面研究單個(gè)或數(shù)個(gè)干細(xì)胞向成骨和成脂方向分化奠定了方法學(xué)的基礎(chǔ)。 2.揭示細(xì)胞形狀影響干細(xì)胞分化,并發(fā)現(xiàn)對(duì)于骨髓基質(zhì)干細(xì)胞分化存在最佳長徑比。利用我們課題組發(fā)展的PEG水凝膠表面微圖案化技術(shù),實(shí)現(xiàn)了骨髓基質(zhì)干細(xì)胞的長時(shí)間定位,并在合適條件下持久維持所設(shè)定的圓形、方形、三角形、星形以及不同長徑比的矩形等一系列不同形狀,揭示細(xì)胞形狀顯著影響干細(xì)胞在成骨/成脂分化液中的分化程度,如：圓形細(xì)胞更易于向成脂肪細(xì)胞分化、星形細(xì)胞更易于向成骨細(xì)胞分化；并且在考察長徑比效應(yīng)時(shí)發(fā)現(xiàn),雖然成脂分化隨長徑比增加單調(diào)下降,但是成骨分化隨著長徑比上升呈現(xiàn)峰值,即,存在最佳長徑比。 3.系統(tǒng)研究了細(xì)胞尺寸、細(xì)胞接觸和細(xì)胞密度對(duì)于干細(xì)胞分化的影響,利用化學(xué)圖案化技術(shù)解構(gòu)了細(xì)胞密度影響干細(xì)胞分化的內(nèi)在物理因素。論文利用了微圖案化技術(shù),一種細(xì)胞接種密度下在不同黏附島上實(shí)現(xiàn)了不同的局部密度,并且可以獨(dú)立研究細(xì)胞尺寸、細(xì)胞之間的接觸以及細(xì)胞密度的影響。結(jié)論證實(shí),細(xì)胞尺寸和接觸都會(huì)影響骨髓基質(zhì)干細(xì)胞的成骨分化和成脂分化,而密度效應(yīng)則是尺寸效應(yīng)和接觸效應(yīng)的綜合結(jié)果。隨著細(xì)胞密度上升,對(duì)于成脂分化,細(xì)胞尺寸效應(yīng)和接觸效應(yīng)給出同樣的趨勢；而對(duì)于成骨分化,細(xì)胞尺寸效應(yīng)和細(xì)胞接觸效應(yīng)在細(xì)胞密度上升時(shí)則給出了“競爭”性的效應(yīng)。我們在利用圖案化技術(shù)解構(gòu)細(xì)胞密度對(duì)干細(xì)胞分化影響的內(nèi)在的物理因素的基礎(chǔ)上,進(jìn)一步提出了影響干細(xì)胞分化的細(xì)胞密度的兩個(gè)區(qū)段的劃分概念。
[Abstract]:The interaction of cells and materials is a basic scientific problem in the study of biological materials, cell biology and regenerative medicine. The relevant research is of great significance to the design of biological materials. The cell, as the basic unit of life activity, can directly reflect the changes of the intrinsic nature of the cell. However, there has been insufficient progress in this regard due to the relative shortage of efficient single-cell research instruments. the micro-pattern technology can realize the cell adhesion contrast on the substrate, so that the positioning of the cells can be controlled, the adhesion of the single cell can be effectively realized through the size of the cell adhesion area, that is, the size of the adhesion island, and the number of the samples can be used for counting and obtaining the credible conclusion, and has good application prospect in the research of single-cell. The tissue is not composed of single cells, but is one of several or more cells and a matrix of extracellular matrix. The interaction between the cells and the cells is also an important basic science in the tissue engineering Problem. The degree of aggregation of multicellular, that is, the cell density is also very high It is important that by controlling the positioning of the cells, the contact of the cells with the cells, the degree of aggregation of the cells, and the like can be effectively controlled by controlling the positioning of the cells, so that the interaction between the multiple cells can be effectively controlled. The bone marrow stromal stem cell has multi-directional differentiation potential and is easy to be amplified in vitro, and the bone marrow stromal stem cells are self-contained and not related to the field of ethics, and are ideal for tissue engineering. Subcell type. Stem cell differentiation is today's white-and-white science This dissertation uses bone marrow stromal stem cells as a model to study the various physical factors that affect the differentiation of stem cells, including the shape effect of single cell differentiation, the size effect, and the dry matter in the multi-cell state. The density effect of cell differentiation. The new work includes the following aspects: 1. Preparation of a strong cell adhesion contrast The micro-patterned surface of the micro-patterned surface is a high-molecular hydrogel, and a criterion for the area of the adhesion island suitable for cell shape study and a microphotograph system are proposed. A semi-quantitative method for determining the degree of differentiation of stem cells was prepared. The micro-pattern of arginine-glycinate (RGD) on the surface of water-gel of polyethylene glycol (PEG) was prepared in the working process, and the cell adhesion was strongly resisted by the polymer. On this basis, a statistical method for the determination of bone differentiation and adipocyte differentiation of bone marrow stromal stem cells was established by using the cell micrographs of the patterned surface, and the most suitable for investigating the shape of the cells was proposed. so as to successfully realize the positioning of single cells with different shapes and length-to-diameter ratios, and reasonably and effectively study the single or several stem cells into the bone and the fibroblast on the basis of the micropatterned surface. the foundation of the methodology was laid down in the direction of the fat, 2. The cell shape was revealed to affect the differentiation of the stem cells and was found to be The best aspect ratio exists in the differentiation of the bone marrow stromal stem cells. The long-time positioning of the bone marrow stromal stem cells is achieved by using the PEG hydrogel surface micropatterning technology developed by our research group, and the set circular, square, triangular, star shape is maintained for a long time under appropriate conditions. and a series of different shapes, such as rectangles of different length-to-length ratios, reveal that the shape of the cells significantly affects the degree of differentiation of the stem cells in the bone/ fat-forming differentiation liquid, such as, the circular cells are more easily differentiated into the fat cells, and the star-shaped cells are more likely to be fine to the bone Cell differentiation; and in the study of the length-to-length ratio, it was found that, although the adipocyte differentiation increased monotonically with the aspect ratio, it was found that the differentiation of the bone along with the length-length ratio The effect of cell size, cell contact and cell density on the differentiation of stem cells was studied by using the chemical patterning technique. The internal physical factors that influence the differentiation of the stem cells by the density of the cells are constructed. The paper makes use of the micropatterning technique, and a different local density is realized on the different adhesion islands under the density of cell inoculation, and the fine pattern can be studied independently. The effects of cell size, cell size, and cell density have been found to affect the differentiation and differentiation of bone marrow stromal stem cells into bone marrow stromal stem cells. The density effect is a comprehensive result of the size effect and the contact effect. As the cell density increases, the same trend is given for adipocyte differentiation, cell size effect and contact effect. The effect of "" on stem cell differentiation is further put forward based on the intrinsic physical factors of the influence of the density of the cells on the differentiation of the stem cells by using the patterning technique.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【共引文獻(xiàn)】

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