二维基质材料和胶体微粒的理化性质对骨髓间充质干细胞分化行为的影响
发布时间：2018-12-15 15:26
【摘要】：随着骨髓间充质干细胞(MSC)在生物医用领域的广泛应用,如何通过改变生物材料的性质来调控MSC的分化成为目前的研究热点。本文一方面研究了理化性质可控的二维材料界面,研究材料表面化学性质和物理模量对MSC分化行为的的调控作用。另一方面,从细胞外微环境过渡到细胞内,研究细胞吞噬纳米微粒对MSC分化行为的影响。选取了广泛用于生物材料界面改性的聚电解质多层膜,利用静电层层自组装的方法制备了氧化石墨烯掺杂聚赖氨酸(PLL)/透明质酸(HA)的多层膜,通过改变氧化石墨烯的位置成功地实现了对多层膜性质的调控,结果发现氧化石墨烯的组装位置越靠近表层,MSC分泌的神经分化相关因子神经巢蛋白和p3-微管蛋白的趋势越明显。为了考察材料模量和可诱导干细胞分化的阿伦膦酸钠分子(Aln)密度双重作用对MSC成骨分化的影响,利用自由基聚合的方法合成了不同模量(4-40 kPa)和阿伦磷酸钠(Aln)密度(0,0.2和4μM)的明胶水凝胶,研究了碱性磷酸酶(ALP),I型胶原(COL)、骨钙蛋白(OCN)以及钙质的表达,发现同时提高模量和Aln密度可以协同促进MSC的成骨分化,且Aln分子和高模量基底对于成骨分化的促进效应在某种程度上是相近的。细胞外微环境与MSC的作用集中于MSC与基质的相互作用,然而吞噬胶体微粒可以直接与细胞作用进而对细胞行为产生影响。考察了牛血清白蛋白包覆的聚乳酸-乙醇酸微粒(PLGA-BSA)对MSC分化的影响。MSC在吞噬PLGA-BSA微粒以后,分泌的ALP活性明显升高,且成骨相关的信号因子COL和OCN在基因和蛋白上的表达均得到了显著的提升,钙质的沉积明显增多；同时成脂相关的过氧化物酶体增殖物激活受体γ(PPARγ)和脂蛋白脂酶(LPL)在基因和蛋白水平上的表达均受到了显著的抑制。说明PLGA-BSA微粒的吞噬可以显著促进MSC的成骨分化,抑制MSC的成脂肪分化。为了考察吞噬微粒后,微粒在细胞内响应对MSC分化的影响,制备了Fe3O4纳米微粒掺杂的、磁性有显著差异的三种BSA微粒(BSA、FB3.4和FB13.6),将上述微粒与MSC共培养24h后外加磁场,考察其对MSC分化行为的调控。MSC在吞噬具有磁性的微粒FB3.4和FB13.6以后,通过施加磁场可以显著的抑制MSC的增殖,促进ALP的分泌,且促进成骨相关的信号因子COL和OCN在基因和蛋白上的表达以及钙质的沉积,说明了MSC吞噬磁性微粒后,在磁场作用下其成骨分化得到了显著的促进与提高。
[Abstract]:With the wide application of bone marrow mesenchymal stem cell (MSC) in biomedical field, how to regulate the differentiation of MSC by changing the properties of biomaterials has become a hot topic. On the one hand, the interface of two-dimensional materials with controllable physical and chemical properties is studied, and the effects of surface chemical properties and physical modulus on the differentiation behavior of MSC are studied. On the other hand, the effect of cell phagocytosis on the differentiation of MSC was studied. Polyelectrolyte multilayers, which are widely used in interfacial modification of biomaterials, are used to prepare polyelectrolyte multilayers of graphene oxide doped polylysine (PLL) / hyaluronic acid (HA) by electrostatic layer self-assembly. By changing the location of graphene oxide, the properties of multilayer films were successfully regulated. It was found that the assembly position of graphene oxide was closer to the surface layer. The trend of nestin and p3-tubulin secreted by MSC was more obvious. In order to investigate the effects of the moduli of materials and the (Aln) density of alendronate molecules that can induce stem cell differentiation on the osteogenic differentiation of MSC. Gelatin hydrogels with different moduli (4-40 kPa) and (Aln) densities of alendronate (0 0. 2 and 4 渭 M) were synthesized by free radical polymerization. The alkaline phosphatase (ALP), I type collagen (COL), was studied. The expression of osteocalcin (OCN) and calcium showed that both moduli and Aln densities could promote the osteogenic differentiation of MSC, and the effects of Aln molecules and high modulus substrates on osteogenic differentiation were similar to each other to some extent. The interaction between extracellular microenvironment and MSC is concentrated on the interaction between MSC and matrix, but phagocytosis of colloidal particles can directly interact with cells and then affect cell behavior. The effect of bovine serum albumin coated polylactic acid-glycolate (PLGA-BSA) on the differentiation of MSC was investigated. The activity of ALP secreted by MSC increased after PLGA-BSA particles were phagocytized. The expression of COL and OCN in genes and proteins were significantly increased, and the deposition of calcium was increased significantly. At the same time, the expression of peroxisome proliferator-activated receptor 纬 (PPAR 纬) and lipoprotein lipase (LPL) were significantly inhibited at both gene and protein levels. The results showed that the phagocytosis of PLGA-BSA particles could significantly promote the osteogenic differentiation of MSC and inhibit the adipogenic differentiation of MSC. In order to investigate the effect of phagocytic particles on the differentiation of MSC, three kinds of BSA particles (BSA,FB3.4 and FB13.6) doped with Fe3O4 nanoparticles were prepared. After co-cultured with MSC for 24 hours, the external magnetic field was applied to investigate the regulation of the differentiation behavior of MSC. After phagocytosis of FB3.4 and FB13.6, MSC could significantly inhibit the proliferation of MSC and promote the secretion of ALP by applying magnetic field. The expression of COL and OCN on genes and proteins and the deposition of calcium showed that MSC phagocytosis of magnetic particles significantly promoted and enhanced the osteogenic differentiation of MSC under the action of magnetic field.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08
本文编号：2380875
[Abstract]:With the wide application of bone marrow mesenchymal stem cell (MSC) in biomedical field, how to regulate the differentiation of MSC by changing the properties of biomaterials has become a hot topic. On the one hand, the interface of two-dimensional materials with controllable physical and chemical properties is studied, and the effects of surface chemical properties and physical modulus on the differentiation behavior of MSC are studied. On the other hand, the effect of cell phagocytosis on the differentiation of MSC was studied. Polyelectrolyte multilayers, which are widely used in interfacial modification of biomaterials, are used to prepare polyelectrolyte multilayers of graphene oxide doped polylysine (PLL) / hyaluronic acid (HA) by electrostatic layer self-assembly. By changing the location of graphene oxide, the properties of multilayer films were successfully regulated. It was found that the assembly position of graphene oxide was closer to the surface layer. The trend of nestin and p3-tubulin secreted by MSC was more obvious. In order to investigate the effects of the moduli of materials and the (Aln) density of alendronate molecules that can induce stem cell differentiation on the osteogenic differentiation of MSC. Gelatin hydrogels with different moduli (4-40 kPa) and (Aln) densities of alendronate (0 0. 2 and 4 渭 M) were synthesized by free radical polymerization. The alkaline phosphatase (ALP), I type collagen (COL), was studied. The expression of osteocalcin (OCN) and calcium showed that both moduli and Aln densities could promote the osteogenic differentiation of MSC, and the effects of Aln molecules and high modulus substrates on osteogenic differentiation were similar to each other to some extent. The interaction between extracellular microenvironment and MSC is concentrated on the interaction between MSC and matrix, but phagocytosis of colloidal particles can directly interact with cells and then affect cell behavior. The effect of bovine serum albumin coated polylactic acid-glycolate (PLGA-BSA) on the differentiation of MSC was investigated. The activity of ALP secreted by MSC increased after PLGA-BSA particles were phagocytized. The expression of COL and OCN in genes and proteins were significantly increased, and the deposition of calcium was increased significantly. At the same time, the expression of peroxisome proliferator-activated receptor 纬 (PPAR 纬) and lipoprotein lipase (LPL) were significantly inhibited at both gene and protein levels. The results showed that the phagocytosis of PLGA-BSA particles could significantly promote the osteogenic differentiation of MSC and inhibit the adipogenic differentiation of MSC. In order to investigate the effect of phagocytic particles on the differentiation of MSC, three kinds of BSA particles (BSA,FB3.4 and FB13.6) doped with Fe3O4 nanoparticles were prepared. After co-cultured with MSC for 24 hours, the external magnetic field was applied to investigate the regulation of the differentiation behavior of MSC. After phagocytosis of FB3.4 and FB13.6, MSC could significantly inhibit the proliferation of MSC and promote the secretion of ALP by applying magnetic field. The expression of COL and OCN on genes and proteins and the deposition of calcium showed that MSC phagocytosis of magnetic particles significantly promoted and enhanced the osteogenic differentiation of MSC under the action of magnetic field.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08
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