生物材料特性及微环境对间充质干细胞向心肌细胞分化的作用研究

发布时间：2018-07-29 12:32

【摘要】：心血管疾病是全世界死亡最主要的原因之一。研究心血管疾病的机理、药物的开发和疾病的诊断治疗需要来自各学科的共同努力。体外构建心肌组织模型将为临床前研究和转化应用提供一个新的手段。体外心肌组织模型是具有人类心肌生理功能的模型,由生物材料和微系统作为组织形成的支架,并且需要人来源的心肌细胞在支架上培养形成具有生理功能的心肌组织。细胞是生命的基本单位,细胞通过粘附在细胞外基质中得以生存和生长。细胞外基质的微结构可以通过改变细胞的形状和细胞骨架来影响细胞的一系列行为,包括细胞的迁移、增殖和分化。人工构建生物支架用于细胞的体外培养和研究,不仅可以模拟细胞外基质为细胞提供支撑,还可以通过调节生物支架微结构来主动影响细胞的行为,比如指导干细胞的定向分化。指导干细胞定向分化是干细胞研究的主要目的之一。本论文的研究目的就是利用生物支架促进成体于细胞向心肌细胞分化,为体外心肌组织形成提供细胞来源。间充质干细胞是研究干细胞定向分化中最常用的细胞。一方面,间充质干细胞具有多向分化潜能,可以分化为成骨细胞、脂肪细胞,心肌细胞和软骨细胞等;另一方面,间充质干细胞的来源广泛,分离和提取的方法简便快捷,并且不涉及伦理问题。本论文选用的干细胞是人脐带中分离提取的间充质干细胞,具有免疫源性低,易于稳定培养扩增的优点。本论文选择明胶作为生物支架材料,明胶是胶原的水解产物,是一种蛋白质,结构中含大量羟基,亲水性极好,制成凝胶时适于细胞培养。通过生物交联剂京尼平的交联可提高明胶的稳定性,获得与体内心肌组织的弹性模量相匹配的水凝胶。利用软刻蚀技术中的微印章方法,以具有微结构的硅片为模板,经聚二甲基硅氧烷(PDMS)转印,成功制备了表面具有微结构的水凝胶,定向排列的微结构可促使细胞高度有序排列。氧化石墨烯(GO)以高比表面积,多羟基,亲水性的物理性能,与明胶水凝胶混合,可提高水凝胶的表面粗糙度,使水凝胶拥有较高的孔隙率,孔径减小,制备的氧化石墨烯水凝胶促进干细胞生长和分化。本论文研究了在玻片、明胶水凝胶、GO掺杂明胶水凝胶、具有微结构的GO掺杂明胶水凝胶这四种生物支架表面利用5-氮胞苷,小分子,或小分子结合共培养三种诱导方法诱导人脐带间充质干细胞(hUCMSCs)分化为心肌细胞。研究结果表明,上述三种明胶类水凝胶都具有很好的细胞相容性,相比于玻片,对人脐带间充质干细胞分化为心肌细胞有显著的促进作用,其中GO掺杂的凝胶作用更明显。相比于使用5-氮胞苷的化学诱导方法,小分子诱导人脐带间充质干细胞分化为心肌细胞的作用更明显,进一步的小分子结合共培养的诱导方法其对hUCMSCs分化为心肌细胞的作用最显著。该结果对干细胞向心肌细胞的高效定向分化研究具有指导意义。
[Abstract]:Cardiovascular disease is one of the leading causes of death in the world. Research on the mechanism of cardiovascular disease, the development of drugs and the diagnosis and treatment of disease require joint efforts from various subjects. The construction of myocardial tissue model in vitro will provide a new hand for preclinical research and transformation. The model of cardiac tissue in vitro is of human heart. A model of the physiological function of the muscle, a scaffold formed by biological materials and Microsystems, and the need for human derived cardiomyocytes to form a physiological function of myocardial tissue on the scaffold. Cells are the basic units of life. Cells have to survive and grow through adhesion to the extracellular matrix. The microstructures of the extracellular matrix can be found. By changing the shape and cytoskeleton of cells to affect a series of behavior of cells, including cell migration, proliferation and differentiation. The artificial construction of scaffolds for the culture and study of cells in vitro can not only simulate the extracellular matrix to provide support for cells, but also affect cell lines by regulating the microstructures of biological scaffolds. For example, directing the directional differentiation of stem cells. Directing stem cell differentiation is one of the main purposes of stem cell research. The purpose of this paper is to use biological scaffolds to promote the differentiation of adult cells into cardiomyocytes and provide a cell source for the formation of cardiac tissue in vitro. Mesenchymal stem cells are the most important research in stem cell differentiation. On the one hand, mesenchymal stem cells have multiple differentiation potential, which can be divided into osteoblasts, adipocytes, cardiomyocytes and chondrocytes. On the other hand, mesenchymal stem cells are widely used, and the methods of isolation and extraction are simple and fast, without ethical problems. The extracted mesenchymal stem cells have the advantages of low immune origin and easy to stable culture and expansion. This paper chooses gelatin as a scaffold material, gelatin is a product of collagen hydrolysate. It is a protein, which contains a large number of hydroxyl groups and excellent hydrophilicity. It is suitable for cell culture when the gel is made. Through cross-linking of biker genipin It can improve the stability of gelatin and obtain hydrogels that match the elastic modulus of myocardial tissue in the body. Using the microseal method in the soft etching technology, the microstructures of the hydrogels on the surface are successfully prepared by using the microstructural silicon chip as the template, and the microstructures of the microstructures are prepared by the transfer of polymethylsiloxane (PDMS). The microstructures of the directional arrangement can promote the cells. Graphene oxide (GO) has high surface area, polyhydroxy, hydrophilic physical properties and mixed with gelatin hydrogel, which can improve the surface roughness of the hydrogel, make the hydrogel have higher porosity, reduce the pore size, and promote the growth and differentiation of the stem cells. Gelatine gel, GO doped gelatin hydrogel and microstructured GO doped gelatin gel, the four kinds of biological scaffolds with 5- azytidine, small molecules, or small molecule binding co culture three induction methods to induce human umbilical cord mesenchymal stem cells (hUCMSCs) to differentiate into cardiomyocytes. The results showed that the above three gelatin hydrogels were all equipped with the above three kinds of gelatin hydrogels. The cytocompatibility of human umbilical cord mesenchymal stem cells significantly promotes the differentiation of human umbilical cord mesenchymal stem cells into cardiomyocytes, and the effect of GO doped gels is more obvious. Compared with the chemical induction of 5- azacytidine, small molecules induce the differentiation of human umbilical cord mesenchymal stem cells into cardiomyocytes. The effect of small molecules combined with co culture on the differentiation of hUCMSCs into cardiomyocytes is most significant. This result is of guiding significance for the study of the efficient and directional differentiation of stem cells to cardiomyocytes.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R318.08

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