基于生物组学的羟基磷灰石骨诱导机理研究

发布时间：2018-07-09 13:34

  本文选题：羟基磷灰石 + 间充质干细胞　； 参考：《东南大学》2015年博士论文

【摘要】：目前,骨损伤修复存在的主要困难之一是可用于填充缺损和促进骨生长的生物材料非常有限。羟基磷灰石是构成骨骼的主要无机成分,由于具有良好的生物相容性、骨传导性和骨诱导性,现已被广泛应用于人体骨组织修复中。羟基磷灰石的主要制备方法包括化学合成(称之为合成羟基磷灰石)和从骨骼和牙齿、鱼鳞等天然生物体内提取(称之为天然羟基磷灰石)两种方式。合成羟基磷灰石是一个化学分子式为Ca10(PO4)6(OH)2的化学计量材料；而天然羟基磷灰石除了羟基磷灰石的成分以外还包含与骨无机成分相似的微量离子,因此具有与骨矿化成分最大的相似性。目前,基于天然与合成羟基磷灰石医用生物材料均已被广泛使用,但对于天然与合成羟基磷灰石的理化性质的对比研究仍然较少,对于两种材料生物相容性和碱性磷酸酶活性等成骨性能的比较更少,更未见采用蛋白质组学等高通量技术比较和研究其成骨诱导机理的报道。本课题组前期对天然与合成羟基磷灰石进行了制备与表征,然后对天然与合成羟基磷灰石的蛋白吸附特性进行了比较,并进一步通过基因表达谱芯片技术结合生物信息学分析研究了天然羟基磷灰石对小鼠骨髓间充质干细胞成骨分化的影响。在前期研究的基础上,本论文将对天然与合成羟基磷灰石的理化性质、生物学性能、骨修复能力及其分子机理进行系统的比较,然后通过对天然羟基磷灰石作用于细胞后的转录组学、蛋白组学和miRNA的联合分析,探讨天然羟基磷灰石诱导骨髓间充质干细胞成骨分化所介导的生物学通路及通路中miRNA的调节作用。本论文包括两个部分的内容,第一部分是天然和合成羟基磷灰石理化性质及生物学性能比较研究,主要内容包括：1)对天然与合成羟基磷灰石粉体和圆盘状试样进行了制备与理化性质的表征,结果显示,与合成HA相比,天然HA中存在特有的碳酸根、磷酸氢根及镁和钠元素；并且天然羟基磷灰石具有更大的单晶尺度和更高的结晶度;此外,浸提液中钙离子的检测表明天然HA可能具有更高的稳定性,而合成HA则具有更高的溶解-沉积特性；镁离子检测表明仅在天然HA组出现镁离子溶出。2)采用贴壁筛选与密度梯度离心分离相结合的方法对小鼠骨髓间充质干细胞进行了分离提取,然后采用流式细胞术进行鉴定了细胞表面标志物和细胞的同质性,结果显示,第4代间充质干细胞高表达细胞粘附因子CD29和CD44,而低表达淋巴细胞标志物CD14和造血细胞标志物CD34,这表明第4代细胞已经得到了纯化,所培养的细胞为骨髓间充质干细胞。3)采用荧光染色和MTT测定方法研究间充质干细胞在天然与合成羟基磷灰石圆盘状试样表面的形貌和增殖情况,结果显示,天然与合成羟基磷灰石表面所培养的细胞形貌相似,与对照组相比材料表面细胞更易呈团聚状生长且增殖缓慢。4)采用基于iTRAQ标记的二维液相色谱串联质谱联用技术对天然与合成羟基磷灰石表面生长细胞的蛋白质表达情况进行研究,在天然与合成羟基磷灰石组24和72小时共鉴定出可信蛋白质800个；对所选蛋白质的表达情况进行Western blot验证显示两种方法所得结果具有较好的一致性,这表明蛋白质组学数据可信。5)对蛋白质数据进行生物信息学分析,结果显示天然与合成羟基磷灰石对细胞主要的GO功能类别影响相似,但在“对无机物质的反应”、“离子跨膜运输”、“生物矿化”和“血管发育”等功能中存在一定的差异；此外,两种材料能够通过特定生物学通路介导细胞的增殖、形貌和分化功能,并且生物学通路之间能够通过蛋白质相互作用网络而相互联系。6)对培养于天然与合成羟基磷灰石表面的细胞进行矿化检测,结果表明两种材料表面细胞的矿化程度均高于对照组,但天然羟基磷灰石能够对矿化作用产生更大的促进作用。第二部分是天然羟基磷灰石成骨诱导机理的转录组学、蛋白质组学和miRNA联合分析,主要内容包括：1)对天然羟基磷灰石对细胞作用的蛋白质组学和基因表达谱芯片数据进行比较,结果显示,两组数据在各时间点的上调表达基因或蛋白质数量均大于下调表达的基因或蛋白质数量；对蛋白质组学数据进行GO功能分析共筛选到4个与成骨分化相关的节点,分别为骨骼发育、细胞分化调控、细胞分化负向调控和细胞分化正向调控。这4个节点包含在基因表达谱芯片数据所涉及的13个与成骨分化相关的节点中。在这4个节点中最终筛选得到10个与成骨分化相关的蛋白质。2)对蛋白质组学数据与基因表达谱芯片数据进行生物学通路联合分析,共得到89条嬖RNA和蛋白质共同参与的生物学通路,其中MAPK信号通路和TGF-β受体信号通路等可能在天然羟基磷灰石所诱导的成骨分化中发挥重要作用。3)对microRNA在天然羟基磷灰石成骨诱导中的调节作用进行分析,结果显示在所检测的13个microRNA中,有9个microRNA在分化相关通路中发挥了调控作用,其中miR-26a和miR-26b可能通过调控成脂分化通路从而对细胞的成脂分化产生抑制作用,而miR-222可能通过调控MAPK通路从而在细胞的成骨分化中发挥重要的促进作用。4)对天然羟基磷灰石的成骨诱导能力进行了检测,碱性磷酸酶染色实验显示培养于天然羟基磷灰石表面的间充质干细胞能够被诱导成骨分化；成骨分化通路验证试验表明,天然羟基磷灰石通过MEK1/2-ERK1/2和JNK MAPK通路共同介导细胞的成骨分化,其中MEK1/2-ERK1/2 MAPK通路在成骨诱导中发挥主要作用。
[Abstract]:Currently, one of the main difficulties in repairing bone damage is that biological materials that can be used to fill defects and promote bone growth are very limited. Hydroxyapatite is the main inorganic component of bone. It has been widely used in the repair of human bone tissue because of its good biocompatibility, bone conductivity and bone inducibility. The main preparation methods of stone include chemical synthesis (called synthetic hydroxyapatite) and two natural organisms (called natural hydroxyapatite) from bone and teeth and fish scale. Synthetic hydroxyapatite is a chemical measurement material of chemical molecular formula Ca10 (PO4) 6 (OH) 2; and natural hydroxyapatite is the addition of hydroxyl group. Apatite also contains trace ions similar to bone mineral components, so it has the greatest similarity with bone mineralized components. At present, natural and synthetic hydroxyapatite medical biomaterials are widely used, but there are still less studies on the physical and chemical properties of natural and synthetic hydroxyapatite. For the two species, the physical and chemical properties of hydroxyapatite are still less. The biomaterial biocompatibility and alkaline phosphatase activity are less than those of the material, and there is no report on the comparison and study of the mechanism of osteogenesis induced by high flux techniques such as proteomics. The effects of natural hydroxyapatite on the osteogenesis of bone marrow mesenchymal stem cells in mice were studied by gene expression spectrum chip technology and bioinformatics analysis. On the basis of previous research, the physical and chemical properties, biological properties and bone repair ability of natural and synthetic hydroxyl phosphite were studied. The force and its molecular mechanism are compared systematically, and then by the combined analysis of natural hydroxyapatite on post cell transcripology, proteomics and miRNA, the biological pathway mediated by natural hydroxyapatite induced bone marrow mesenchymal stem cells differentiation and the regulation of miRNA in the pathway of bone marrow mesenchymal stem cells are discussed. This paper includes two The first part is a comparative study of physicochemical properties and biological properties of natural and synthetic hydroxyapatite. The main contents are as follows: 1) the preparation and physicochemical properties of natural and synthetic hydroxyapatite powders and disc like samples are characterized. The results show that there are special carbonates and phosphoric acid in natural HA compared with the synthetic HA. Hydrogen roots and magnesium and sodium, and the natural hydroxyapatite has a larger single crystal scale and higher crystallinity; in addition, the detection of calcium ions in the extract indicates that the natural HA may have higher stability, while the synthetic HA has a higher dissolution deposition characteristic; magnesium ion test shows that only the magnesium ion dissolves.2 in the natural HA group. The mouse bone marrow mesenchymal stem cells were isolated and extracted by the combination of adherent screening and density gradient centrifugation. Then the cell surface markers and cell homogeneity were identified by flow cytometry. The results showed that the fourth generations of mesenchymal stem cells were high in cell adhesion factor CD29 and CD44, while low expression lymph nodes were expressed. Cell marker CD14 and hematopoietic cell marker CD34, which indicate that the fourth generation cells have been purified, the cultured cells are bone marrow mesenchymal stem cells (.3), and the morphology and proliferation of mesenchymal stem cells on the surface of natural and synthetic hydroxyapatite disk like specimens are studied by fluorescence staining and MTT assay. The morphology of the cells cultured on the surface of synthetic hydroxyapatite was similar. Compared with the control group, the surface cells of the material were more likely to be agglomerated and proliferated slowly.4). The protein expression of natural and synthetic hydroxyapatite surface long cells was studied by the two-dimensional liquid chromatography tandem mass spectrometry based on iTRAQ markers. A total of 800 trusted proteins were identified by natural and synthetic hydroxyapatite 24 and 72 hours. Western blot verification of the expression of the selected proteins showed that the results obtained by the two methods had good consistency, which showed that the proteomic data trusted.5) for bioinformatics analysis of protein data, and the results showed that the protein was natural. The effects of synthetic hydroxyapatite on the main GO functional categories of cells are similar, but there are certain differences in the functions of "reaction to inorganic substances", "ion transmembrane transport", "biomineralization" and "vascular development". In addition, two materials can mediate cell proliferation, morphology and differentiation through a specific biotic pathway. It is possible that the mineralization of cells cultured on the surface of natural and synthetic hydroxyapatite can be mineralized by the interaction of protein interaction network (.6) between biological pathways. The results show that the mineralization of the surface cells on the surface of the two materials is higher than that of the control group, but the natural hydroxyl apatite can produce a greater promotion of mineralization. The second part is the transcriptional histology of the bone induction mechanism of natural hydroxyapatite, proteomics and miRNA combined analysis. The main contents include: 1) the comparison of the proteomics and gene expression chip data of the natural hydroxyapatite to the cells shows that the up-regulated groups of the two groups of data at each time point are up. The number of proteins and proteins were larger than those of down regulated genes or proteins; the GO functional analysis of proteomics data screened 4 nodes related to osteogenesis differentiation, which were bone development, cell differentiation regulation, negative regulation of cell differentiation and positive regulation of cell differentiation. These 4 nodes were included in gene expression chip. Among the 13 nodes associated with osteogenesis, 10 proteins.2 associated with osteogenesis were screened in the 4 nodes. The biological pathway of the proteomic data and gene expression chip data was combined to obtain a total of 89 biological pathways involved in RNA and egg white matter, including the MAPK letter. Signal pathway and TGF- beta receptor signaling pathway may play an important role in the osteogenic differentiation induced by natural hydroxyapatite (.3). The regulation of microRNA in the induction of bone induction in natural hydroxyapatite was analyzed. The results showed that in the 13 detected microRNA, 9 microRNA played a regulatory role in the differentiation related pathway. MiR-26a and miR-26b may inhibit the lipid differentiation of cells by regulating the lipid differentiation pathway, and miR-222 may play an important role in promoting the osteogenic differentiation of the cells by regulating the MAPK pathway.) the osteogenic induction ability of natural hydroxyapatite was detected, and the alkaline phosphatase staining test was used. It is shown that mesenchymal stem cells cultured on the surface of natural hydroxyapatite can be induced to osteogenesis, and the test of osteogenic differentiation pathway shows that the natural hydroxyapatite mediates the osteogenic differentiation of the cells through MEK1/2-ERK1/2 and JNK MAPK pathway, and the MEK1/2-ERK1/2 MAPK pathway plays a major role in the osteogenesis induction.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R68;R318.08
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本文编号：2109530