發(fā)布時(shí)間：2018-12-15 15:26

【摘要】：隨著骨髓間充質(zhì)干細(xì)胞(MSC)在生物醫(yī)用領(lǐng)域的廣泛應(yīng)用,如何通過改變生物材料的性質(zhì)來調(diào)控MSC的分化成為目前的研究熱點(diǎn)。本文一方面研究了理化性質(zhì)可控的二維材料界面,研究材料表面化學(xué)性質(zhì)和物理模量對MSC分化行為的的調(diào)控作用。另一方面,從細(xì)胞外微環(huán)境過渡到細(xì)胞內(nèi),研究細(xì)胞吞噬納米微粒對MSC分化行為的影響。選取了廣泛用于生物材料界面改性的聚電解質(zhì)多層膜,利用靜電層層自組裝的方法制備了氧化石墨烯摻雜聚賴氨酸(PLL)/透明質(zhì)酸(HA)的多層膜,通過改變氧化石墨烯的位置成功地實(shí)現(xiàn)了對多層膜性質(zhì)的調(diào)控,結(jié)果發(fā)現(xiàn)氧化石墨烯的組裝位置越靠近表層,MSC分泌的神經(jīng)分化相關(guān)因子神經(jīng)巢蛋白和p3-微管蛋白的趨勢越明顯。為了考察材料模量和可誘導(dǎo)干細(xì)胞分化的阿倫膦酸鈉分子(Aln)密度雙重作用對MSC成骨分化的影響,利用自由基聚合的方法合成了不同模量(4-40 kPa)和阿倫磷酸鈉(Aln)密度(0,0.2和4μM)的明膠水凝膠,研究了堿性磷酸酶(ALP),I型膠原(COL)、骨鈣蛋白(OCN)以及鈣質(zhì)的表達(dá),發(fā)現(xiàn)同時(shí)提高模量和Aln密度可以協(xié)同促進(jìn)MSC的成骨分化,且Aln分子和高模量基底對于成骨分化的促進(jìn)效應(yīng)在某種程度上是相近的。細(xì)胞外微環(huán)境與MSC的作用集中于MSC與基質(zhì)的相互作用,然而吞噬膠體微�？梢灾苯优c細(xì)胞作用進(jìn)而對細(xì)胞行為產(chǎn)生影響�？疾炝伺Ｑ灏椎鞍装驳木廴樗�-乙醇酸微粒(PLGA-BSA)對MSC分化的影響。MSC在吞噬PLGA-BSA微粒以后,分泌的ALP活性明顯升高,且成骨相關(guān)的信號因子COL和OCN在基因和蛋白上的表達(dá)均得到了顯著的提升,鈣質(zhì)的沉積明顯增多；同時(shí)成脂相關(guān)的過氧化物酶體增殖物激活受體γ(PPARγ)和脂蛋白脂酶(LPL)在基因和蛋白水平上的表達(dá)均受到了顯著的抑制。說明PLGA-BSA微粒的吞噬可以顯著促進(jìn)MSC的成骨分化,抑制MSC的成脂肪分化。為了考察吞噬微粒后,微粒在細(xì)胞內(nèi)響應(yīng)對MSC分化的影響,制備了Fe3O4納米微粒摻雜的、磁性有顯著差異的三種BSA微粒(BSA、FB3.4和FB13.6),將上述微粒與MSC共培養(yǎng)24h后外加磁場,考察其對MSC分化行為的調(diào)控。MSC在吞噬具有磁性的微粒FB3.4和FB13.6以后,通過施加磁場可以顯著的抑制MSC的增殖,促進(jìn)ALP的分泌,且促進(jìn)成骨相關(guān)的信號因子COL和OCN在基因和蛋白上的表達(dá)以及鈣質(zhì)的沉積,說明了MSC吞噬磁性微粒后,在磁場作用下其成骨分化得到了顯著的促進(jìn)與提高。
[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 緯 (PPAR 緯) 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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R318.08

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