HMGB1调控间充质干细胞生物学特性及机制研究

发布时间：2018-02-14 08:48

本文关键词： 高迁移率族蛋白1 间充质干细胞 MIR210 MC3T3-E1 迁移分化 MAPK　出处：《中国人民解放军军事医学科学院》2008年硕士论文　论文类型：学位论文

【摘要】： 人体内的骨髓间充质干细胞(mesenchymal stem cells, MSCs)是启动组织修复的重要干细胞之一。多种趋化因子和组织损伤信号可以调控MSC,并在特定条件下诱导其分化为构成结缔组织的细胞。高迁移效率蛋白1 (High mobility group box 1, HMGB1)被归为一类新的具有细胞趋化作用的组织修复信号分子,可以促进多种细胞的增殖、迁移、分化以及细胞骨架重建和伤口愈合。但该组织修复因子对间充质干细胞生物学特性及机制并不清楚。小鼠细胞系MC3T3-E1是一种类MSC细胞,可以向成骨及脂肪方向分化,并且具有类似的表面标记分子。本研究用人MSC和MC3T3细胞为模型,观察了HMGB1对MSC细胞生物学特性影响的可能机制。我们实验室的前期工作证明人骨髓来源的MSC表达HMGB1受体RAGE和TLR-4,HMBG1能够通过RAGE诱导MSC向成骨细胞分化。本研究鉴定了小鼠细胞系MC3T3-E1的免疫表型及向成骨及脂肪分化的能力。用RT-PCR方法证实MC3T3-E1表达HMGB1的受体RAGE和TLR-2、3、4、9。以该细胞为模型,系统研究了HMGB1对MC3T3-E1促迁移效应及对分化作用的影响。主要结果如下:(1)MC3T3-E1高表达CD29(β1-integrin)、H-2Kb、Sca1及CD34等表面标志分子,而不表达c-Kit(CD117)。(2)在一定的浓度范围(25~100ng/ml)内,HMGB1对MC3T3-E1的增殖基本无影响;但HMGB1对MC3T3有促迁移作用,在25ng/ml浓度时最明显,之后随着浓度的增高,其促迁移作用逐渐减弱。HMGB1能够诱导MC3T3-E1细胞MAPK的活化,而且其诱导的MC3T3-E1细胞的迁移作用依赖于MAPK通路中ERK的活化。另外,在小于25ng/ml的范围内,HMGB1可以升高MC3T3-E1的ALP活性,这与它对MSC的作用是一致的,但超过50ng/ml,HMGB1即表现为抑制MC3T3-E1的ALP活性。HMGB1不能诱导MC3T3-E1向脂肪细胞分化。除了多种细胞信号介导细胞因子的生物学效应外,MicroRNA也被证明是重要的细胞生物学效应的调节分子。MIR210是新近发现的一种小RNA分子,它的作用、结合分子、生物学效应等都尚不清楚。我们前期工作表明HMGB1作用后的细胞中MIR210明显上调,但其对MSC的调节作用并不清楚。利用实时定量PCR的方法观察到在HMGB1作用后MC3T3-E1中明显上调。利用重组慢病毒介导的基因转移方法,将MIR210转入MC3T3-E1细胞,分选转基因细胞并进一步进行生物学效应研究。高表达MIR210对MC3T3-E1及MSC的增殖基本无影响,也不影响MC3T3-E1的表型。但高表达MIR210能够促进MC3T3-E1细胞自发迁移率,以及对HMGB1诱导迁移的敏感性。在应用标准剂量和1/10剂量诱导剂的情况下,MIR210使得MSC及MC3T3-E1的ALP活性降低,提示有可能对这MSC的成骨分化有抑制作用。研究结果表明HMGB1能够诱导MC3T3-E1的分化并通过MAPK诱导其迁移;小RNA MIR210参与了HMGB1生物学效应的调节。该研究深入MSC的调控机制,并为进一步拓展MSC在组织修复中的应用提供一定的理论基础。
[Abstract]:Bone marrow mesenchymal stem cells (MSCs) are one of the important stem cells to initiate tissue repair. Many chemokines and tissue damage signals can regulate MSCs and induce them to differentiate into connective tissue under certain conditions. Cells. High mobility group box 1 (HMGB1) is classified as a new cell chemotactic tissue repair signaling molecule. It can promote the proliferation, migration, differentiation, cytoskeleton reconstruction and wound healing of many kinds of cells. However, the biological characteristics and mechanism of the tissue repair factor for mesenchymal stem cells are not clear. Mouse MC3T3-E1 cell line is a kind of MSC cell line. Human MSC and MC3T3 cells were used as models to observe the possible mechanism of the effect of HMGB1 on the biological characteristics of MSC cells. The previous work in our laboratory proved that HMGB1 receptor RAGE and TLR-4 HMBG1 could induce the differentiation of MSC into osteoblasts by RAGE. The immunophenotype, osteogenesis and adipose differentiation of mouse MC3T3-E1 cell line were identified. RT-PCR method was used to confirm the expression of HMGB1 receptor RAGE and TLR-2O3H4 by MC3T3-E1. The effects of HMGB1 on the migration and differentiation of MC3T3-E1 were systematically studied. The main results were as follows: (1) MC3T3-E1 highly expressed CD29 (尾 _ 1-integrin (尾 _ 1-integrin) H _ 2KbSca1, CD34 and other surface marker molecules, but did not express c-Kitt ~ (1) CD _ (117) 路L ~ (2)). HMGB1 had no effect on the proliferation of MC3T3-E1 in a certain concentration range of 25100ng / ml. However, HMGB1 could promote the migration of MC3T3, which was most obvious at the concentration of 25ng / ml, and then decreased with the increase of concentration. HMGB1 could induce the activation of MAPK in MC3T3-E1 cells. Moreover, the migration of MC3T3-E1 cells induced by HMGB1 depends on the activation of ERK in the MAPK pathway. In addition, HMGB1 can increase the ALP activity of MC3T3-E1 in a range of less than 25 ng / ml, which is consistent with the effect of HMGB1 on MSC. But more than 50 ng / ml HMGB1 could inhibit the ALP activity of MC3T3-E1. HMGB1 could not induce MC3T3-E1 to differentiate into adipocytes. In addition to the biological effects of cytokines mediated by a variety of cellular signals, microRNAs have also been shown to be important modulators of cellular biological effects. MIR210 is a newly discovered small RNA molecule that acts as a binding molecule. The biological effects are still unknown. Our previous work showed that MIR210 was up-regulated in the cells treated with HMGB1. However, its regulatory effect on MSC was not clear. It was observed by real-time quantitative PCR that MC3T3-E1 was up-regulated after HMGB1 treatment. MIR210 was transferred into MC3T3-E1 cells by gene transfer mediated by recombinant lentivirus. The overexpression of MIR210 had no effect on the proliferation of MC3T3-E1 and MSC, nor did it affect the phenotype of MC3T3-E1. However, high expression of MIR210 could promote the spontaneous migration of MC3T3-E1 cells. Under the condition of standard dose and 1/10 dose of inducer, the ALP activity of MSC and MC3T3-E1 was decreased, which suggested that it might inhibit the osteogenic differentiation of MSC. The results showed that HMGB1 could induce the differentiation of MC3T3-E1 and induce its migration through MAPK, and that small RNA MIR210 was involved in the regulation of biological effects of HMGB1. It also provides a theoretical basis for further expanding the application of MSC in tissue repair.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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