材料调控脂肪干细胞命运及其血管生成活性的研究

发布时间：2019-03-21 16:02

【摘要】：在组织工程和再生医学中，通过生物材料模拟天然干细胞微环境，研究材料与干细胞之间的相互作用，是了解体内干细胞行为命运调控方式的主要手段，也是开发新型生物材料的基础。同时，在基于生物材料的干细胞移植治疗中，材料内部的快速血管生成是保持干细胞活性和功能以及治疗效果的必要条件。因此，研究生物材料与干细胞之间的相互作用以及生物材料的血管生成活性，是目前组织工程中一个非常重要的研究方向。本文采用脂肪干细胞作为成体干细胞模型，，研究生物材料与脂肪干细胞的相互作用。首先立足于硫醇自组装单分子膜（SAM）对复杂天然干细胞微环境的化学信号进行解析，研究单一化学信号对于脂肪干细胞命运的调控作用。接下来通过分子自组装方法和静电纺丝技术分别制备出各向同性与各向异性的纳米纤维水凝胶材料：功能化RADA16-I自组装多肽和定向纤维蛋白（Fibrin）。从物理结构上高度模拟干细胞微环境，以实现对干细胞微环境的重构。在表面化学基团与脂肪干细胞相互作用的研究中，发现化学基团在无诱导剂的条件下能够调控脂肪干细胞行为：初期黏附、以及后期形貌、增殖和分化。在此发现的基础上，生物材料对于脂肪干细胞命运的调控因素可以缩小到分子-化学基团水平。通过在RADA16-I上接枝模拟细胞外基质（ECM）蛋白的短肽片断赋予了材料不同的生物学功能，研究了脂肪干细胞在功能化自组装多肽中原位三维培养条件下与不同生物学信号的相互作用，包括细胞迁移、增殖以及生长因子的分泌。通过三维微球模型和鸡胚绒毛尿囊膜（CAM）模型评价了功能化自组装多肽的体内和体外血管生成活性。发现功能化自组装多肽在为内皮细胞创建良好三维微环境的同时可以促进内皮细胞迁移，并提高内皮细胞对于VEGF的响应促进内皮细胞萌芽。CAM体内结果显示功能化自组装多肽具有良好的体内生物相容性和血管生成活性。首次通过静电纺丝技术制备出基于天然生物材料的定向纳米纤维水凝胶，并实现了生物材料/细胞的原位三维培养。观察到定向Fibrin水凝胶通过接触诱导方式可使脂肪干细胞取向成长。Fibrin水凝胶表现出良好的体外血管生物活性，在VEGF作用下可促使前体内皮细胞向成熟内皮细胞分化，并分泌一型胶原、四型胶原、粘连蛋白（laminin）等内皮细胞ECM蛋白。
[Abstract]:In tissue engineering and regenerative medicine, simulating natural stem cell microenvironment by biomaterials and studying the interaction between materials and stem cells is the main means of understanding the behavior and fate of stem cells in vivo. It is also the basis for the development of new biomaterials. At the same time, rapid angiogenesis in biomaterial-based stem cell transplantation is a necessary condition to maintain stem cell activity and function as well as therapeutic effect. Therefore, the research on the interaction between biomaterials and stem cells and the angiogenesis activity of biomaterials is a very important research direction in tissue engineering. Adipose-derived stem cells (ADSCs) were used as adult stem cells to study the interaction between biomaterials and adipose-derived stem cells. Firstly, based on the analysis of the chemical signals of complex natural stem cell microenvironment by mercaptan self-assembled monolayer (SAM), the effects of single chemical signal on the fate of adipose stem cells (ADSCs) were studied. Then isotropic and anisotropic nanofiber hydrogels were prepared by molecular self-assembly and electrospinning, respectively: functionalized RADA16-I self-assembled polypeptide and oriented fibrin (Fibrin). The stem cell microenvironment was simulated from the physical structure to realize the reconstruction of stem cell microenvironment. In the study of the interaction between the surface chemical groups and adipose-derived stem cells, it was found that the chemical groups could regulate the behavior of adipose-derived stem cells without inducer: initial adhesion, as well as morphology, proliferation and differentiation of adipose-derived stem cells. On the basis of these findings, the regulatory factors of biomaterials on the fate of adipose-derived stem cells can be reduced to the level of molecular-chemical groups. The biomaterials were endowed with different biological functions by grafting the short peptide fragments of the extracellular matrix (ECM) protein onto the RADA16-I. The interaction of adipose-derived stem cells (ADSCs) with different biological signals, including cell migration, proliferation and the secretion of growth factors, was studied under the condition of in situ three-dimensional culture of functional self-assembled peptides. Three-dimensional microsphere model and chicken embryo chorioallantoic membrane (CAM) model were used to evaluate the in vivo and in vitro angiogenesis activity of the functional self-assembled polypeptide. It was found that functional self-assembled peptides can promote the migration of endothelial cells while creating a good three-dimensional microenvironment for endothelial cells. Cam in vivo showed that the functionalized self-assembled peptide had good biocompatibility and angiogenesis activity in vivo. Oriented nanofiber hydrogels based on natural biomaterials were prepared by electrospinning for the first time and in situ three-dimensional culture of biomaterials / cells was realized. It was observed that the directional Fibrin hydrogel could induce the growth of adipose stem cells by contact induction. Fibrin hydrogel showed good vascular activity in vitro and induced the differentiation of forebody endothelial cells into mature endothelial cells under the effect of VEGF. ECM protein of endothelial cells such as type I collagen, type 4 collagen and adhesion protein (laminin) were also secreted.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08

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