纳米银及载银表面与人骨髓间充质干细胞的相容性研究

发布时间：2018-05-27 12:40

本文选题：纳米银 + 载银表面　；参考：《清华大学》2016年博士论文

【摘要】：纳米银具有广谱抗菌性,是纳米医药领域使用最广泛的产品之一。它被用于多种医疗器材中,包括创伤敷料、导液管、骨水泥和人工心脏瓣膜等。在组织工程中,纳米银常被用作抗菌剂添加到载有干细胞的支架材料中。因此,迫切需要了解清楚纳米银与干细胞的相容性,以使纳米银更安全地应用于组织工程领域。在本文中,采用多元醇还原法制备了尺寸为30 nm的银颗粒。将纳米银以不同浓度添加到细胞培养液中与细胞共培养,研究其与人骨髓间充质干细胞(hMSCs)的相容性。纳米银能够被hMSCs吞噬,进入到细胞质基质中。高浓度的纳米银能降低细胞的存活率和细胞线粒体膜电位,同时导致细胞释放乳酸脱氢酶和生成过量活性氧(ROS)。纳米银所引发的细胞死亡方式既有凋亡又有坏死。高浓度的纳米银还能导致细胞产生G2/M期阻滞,证明纳米银对细胞的DNA造成了损伤。研究了低浓度的纳米银对hMSCs分化的影响规律。成骨分化结果显示,纳米银促进hMSCs成骨分化相关蛋白的表达,包括碱性磷酸酶(ALP)、I型胶原(COLI)、骨桥蛋白(OPN)和骨钙素(OCN)。此外,纳米银还能促进细胞矿化结的生成,表明纳米银能提高hMSCs的成骨分化能力。成脂分化的研究结果表明,纳米银不影响hMSCs脂联素的分泌、脂滴的生成和成脂分化相关基因的表达。纳米银对hMSCs的成脂分化没有影响。关于成软骨分化,低浓度纳米银长期作用于分化中的细胞,可以促进糖胺聚糖(GAG)和聚集蛋白聚糖的分泌,促进SOX9、软骨寡聚基质蛋白(COMP)基因的表达。而较高浓度纳米银短期作用,能促进SOX9基因的表达,不影响GAG、聚集蛋白聚糖、II型胶原和COMP的表达。两种处理方式都能抑制十型胶原的表达。表明纳米银对hMSCs成软骨分化是有利的,它可以促进成软骨相关基因和蛋白的表达,抑制软骨细胞肥大化。采用等离子体聚合的方法,以烯丙胺为单体,制备了一种富含氨基的表面。再将这种基底浸泡在2-巯基琥珀酸(MSA)包覆的纳米银溶液中8 h,得到了一种覆载纳米银的表面。这种表面具有良好的抗菌性能,并且不影响hMSCs的贴附和增殖。对细胞分化的研究结果表明载银表面不影响hMSCs的成骨分化能力。载银表面能促进hMSCs成脂分化相关基因和蛋白的表达,促进脂滴的形成,从而提高hMSCs的成脂分化能力。载银表面能够促进细胞生成ROS,进而引发细胞内抗氧化酶的表达,这可能是其能够促进hMSCs成脂分化的机理。
[Abstract]:Nano-silver is one of the most widely used products in the field of nano-medicine because of its wide-spectrum antibacterial properties. It is used in a variety of medical devices, including wound dressing, fluid conduit, bone cement and artificial heart valves. In tissue engineering, silver nanoparticles are often used as antimicrobial agents in scaffolds containing stem cells. Therefore, it is urgent to understand the compatibility of silver nanoparticles with stem cells in order to make silver nanoparticles more safely used in tissue engineering. In this paper, 30 nm silver particles were prepared by polyol reduction method. The compatibility of silver nanoparticles with human bone marrow mesenchymal stem cells (hMSCs) was studied by co-culture with different concentrations of silver in cell culture medium. Silver nanoparticles can be swallowed by hMSCs and enter the cytoplasmic matrix. High concentration of silver nanoparticles can reduce cell survival rate and mitochondrial membrane potential, at the same time lead to the release of lactate dehydrogenase and the formation of excessive reactive oxygen species. The cell death induced by nano-silver has both apoptosis and necrosis. High concentration of silver nanoparticles can also cause G 2 / M phase arrest of cells, suggesting that silver nanoparticles damage the DNA of cells. The effect of low concentration of silver nanoparticles on the differentiation of hMSCs was studied. The results of osteogenic differentiation showed that nano-silver promoted the expression of osteogenic differentiation related proteins in hMSCs, including alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN). In addition, nano-silver can also promote the formation of mineralized junctions, indicating that nano-silver can improve the osteogenic differentiation ability of hMSCs. The results of adipogenic differentiation showed that silver nanoparticles did not affect the secretion of adiponectin, the formation of lipid droplets and the expression of genes related to adipogenic differentiation. Silver nanoparticles had no effect on the adipogenic differentiation of hMSCs. As for cartilage differentiation, low concentration of silver nanoparticles can promote the secretion of glycosaminoglycan (GAG) and aggregate proteoglycan, and promote the expression of SOX9, cartilage oligomeric matrix protein (omp) gene. However, high concentration of silver nanoparticles could promote the expression of SOX9 gene in a short time, without affecting the expression of gag, type II collagen and COMP. Both treatments could inhibit the expression of type 10 collagen. The results indicated that silver nanoparticles were beneficial to the differentiation of hMSCs chondrocytes, which could promote the expression of chondrogenic genes and proteins and inhibit the hypertrophy of chondrocytes. An amino-rich surface was prepared by plasma polymerization with allylamine as monomer. Then the substrate was immersed in 2-mercaptosuccinic acid (MSA) coated nano-silver solution for 8 h to obtain a surface coated with nano-silver. This surface has good antibacterial properties and does not affect the adhesion and proliferation of hMSCs. The results of cell differentiation showed that the surface of silver carrier did not affect the osteogenic differentiation ability of hMSCs. Silver-loaded surface can promote the expression of genes and proteins associated with adipogenic differentiation of hMSCs, promote the formation of lipid droplets, and thus improve the ability of lipid differentiation of hMSCs. The surface of silver carrier can promote the formation of ROSs and induce the expression of antioxidant enzymes in the cells, which may be the mechanism of promoting the adipogenic differentiation of hMSCs.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;TB383.1

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