HMGB1的原核表达以及对人骨髓来源的间充质干细胞生物学特性的作用研究

发布时间：2018-04-06 00:09

本文选题：高迁移率族蛋白1　切入点：间充质干细胞　出处：《中国人民解放军军事医学科学院》2006年博士论文

【摘要】：HMGB1可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，因此被归为一类新的具有细胞趋化作用的组织修复信号分子。人体内的MSC是无组织特异性的组织修复启动细胞，多种趋化因子和组织损伤信号分子可以动员MSC，并在特定条件下诱导其分化为构成结缔组织的细胞。我们推测HMGB1很可能会影响MSC的迁移、增殖和分化等生物学特性。因此，本研究观察了HMGB1对MSC表型、增殖、迁移，以及向成骨、成软骨和成脂肪分化性能的影响及其相关机制。我们通过RT-PCR方法扩增出人HMGB1的全长编码基因，克隆于原核表达载体pET-24a-d(+)，从而构建了HMGB1的原核表达载体；经IPTG诱导表达，通过亲和层析方法纯化得到了HMGB1融合蛋白。该蛋白在体外能够诱导大鼠血管平滑肌细胞的迁移，，证明具有生物学活性。我们从人骨髓中分离并培养了MSC，鉴定了其免疫表型和多向分化潜能。RT-PCR方法证实MSC表达HMGB1的受体RAGE和TLR-4。在此基础上较系统研究了HMGB1对MSC生物学特性的影响。 1．HMGB1对MSC免疫表型的影响。25ng／ml的HMGB1作用2周后，MSC的免疫表型CD29、CD166、CD73、HLA-DR、CD80和CD86的表达均未发生明显变化，提示此浓度的HMGB1不影响其免疫表型。 2．HMGB1对MSC向成骨细胞分化特性的影响。HMGB1可提高MSC的ALP活性，且上调向成骨细胞分化的标志基因和转录因子(OPN、OCN、ALP、runx2)的表达。但并不能体外诱导MSC形成骨结节以及随后的矿化。因此，HMGB1虽然能促进骨髓MSC向成骨细胞分化，但不能诱导MSC分化为成熟的骨细胞。
[Abstract]:HMGB1 can promote the proliferation, migration, differentiation, cytoskeleton reconstruction and wound healing of many kinds of cells, so it is classified as a new cell chemotactic tissue repair signal molecule.MSC in human body is a non-tissue specific tissue repair primer. A variety of chemokines and tissue damage signaling molecules can mobilize MSC and induce it to differentiate into connective tissue cells under specific conditions.We speculate that HMGB1 may affect the biological characteristics of MSC, such as migration, proliferation and differentiation.Therefore, the effects of HMGB1 on the phenotype, proliferation, migration, osteogenesis, cartilage and adipogenic differentiation of MSC and its related mechanisms were investigated.The full-length encoding gene of human HMGB1 was amplified by RT-PCR and cloned into the prokaryotic expression vector pET-24a-d.The prokaryotic expression vector of HMGB1 was constructed, and the HMGB1 fusion protein was purified by affinity chromatography and induced by IPTG.This protein can induce the migration of rat vascular smooth muscle cells in vitro, which proves that it has biological activity.We isolated and cultured MSCs from human bone marrow and identified their immunophenotype and multidirectional differentiation potential. RT-PCR confirmed the expression of HMGB1 receptors RAGE and TLR-4 by MSC.On this basis, the effects of HMGB1 on the biological characteristics of MSC were systematically studied.The effect of 1.HMGB1 on the immunophenotype of MSC. The expression of CD29, CD166, CD76, HLA-DRN, CD80 and CD86 did not change significantly after two weeks of HMGB1 treatment with 1.HMGB1, suggesting that this concentration of HMGB1 did not affect the immunophenotype of MSC.Effect of 2.HMGB1 on the differentiation of MSC into osteoblasts. HMGB1 increased the ALP activity of MSC and up-regulated the expression of osteoblast differentiation marker gene and transcription factor (OPN).However, the formation of bone nodules and subsequent mineralization of MSC could not be induced in vitro.HMGB1 can promote the differentiation of bone marrow MSC into osteoblasts, but it can not induce MSC to differentiate into mature bone cells.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R341

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