【作者】 张志伟；

【导师】 吕晓迎；

【作者基本信息】 东南大学 ， 生物医学工程， 2015， 博士

【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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。更多还原

【Abstract】 Currently, one of the main difficulties in repairing bone defects is the limited availability of suitable biomaterials capable of filling the defect and promoting bone growth. Hydroxyapatite (HA), the main inorganic component of bone, has been widely used in human bone tissue restoration due to its good biocompatibility, osteoconductive performance and osteoinductive properties. There are two main methods of obtaining HA:synthesis by chemical methods, the product of which is defined as synthetic hydroxyapatite (SHA); extraction from natural tissues such as bone, teeth and scales, the product of which is defined as natural hydroxyapatite (NHA). Synthetic hydroxyapatite is a stoichiometric HA with a chemical composition of Ca10(PO4)6(OH)2. Whereas, besides the composition of HA, natural hydroxyapatite also includes trace amounts of ions which reside in the inorganic component of bone, thus could achieve maximum similarity with natural bone mineral. Nowadays, NHA-and SHA-based medical biomaterials are widely used in bone repair. However, comparative studies of the physicochemical and biological properties of NHA and SHA are still lacking. In particular, high-throughput techniques such as proteomics technique have not yet to be used to compare the bone-repair properties of NHA and SHA.In our group’s previous study, natural and synthetic hydroxyapatites were prepared and characterized, and the protein adsorptive properties of NHA and SHA were compared. In addition, NHA-induced osteogenic differentiation of mesenchymal stem cells (MSCs) was studied using transcriptomic technology combined with bioinformatics analysis. Based on our group’s previous research, the present study will compare systematically natural and synthetic hydroxyapatite, including physicochemical and biological properties, the ability to promote bone repair and their mechanism. Then the NHA-induced osteogenic differentiation pathway and the regulatory function of miRNA in those pathways were studied through an integrated study using transcriptomics, proteomics and miRNA analyses. This work consists of two parts.The first part is the comparative study of physicochemical and biological properties of natural and synthetic hydroxyapatite. The main contents are as follows.1) The powdered and disc-shaped samples of both NHA and SHA were prepared, and the physicochemical properties of the samples were characterized. The results revealed that NHA, rather than SHA, contains carbonate and hydrogen phosphate groups, as well as elements such as Mg and Na. NHA exhibited larger single crystal size and higher crystallinity than SHA. In addition, the detection of Ca ion in the extracts implied that NHA exhibits greater physical stability and that SHA might display higher dissolution-deposition. The detection of Mg ion in the extracts implied Mg ion was released only from NHA.2) Mouse bone marrow derived MSCs were isolated using density gradient centrifugation and adherence screening methods. Then flow cytometric analysis was performed to evaluate cell surface markers and MSCs homogeneity. The results showed that the tested fourth-passage cell samples typically expressed the cell adhesion molecules CD29 and CD44 but were negative for the typical lymphocytic marker CD 14 and hematopoietic marker CD34. These results suggested that fourth-passage MSCs were well-purified, and the cultured cells were indeed MSCs.3) The morphology and proliferation of MSCs cultured on both NHA and SHA were investigated using fluorescent staining and MTT assay. The results showed that MSCs cultured on NHA and SHA displayed similar morphology. Compared with the negative control, cells cultured on both NHA and SHA tended to aggregate during growth and proliferation rates were lower.4) The protein samples of cells cultured on both NHA and SHA were labeled with iTRAQ and then subjected to proteomics using 2-D strong cationic exchange/reversed phase liquid chromatography matrix-assisted laser desorption/ionization-tandem mass spectrometry. The results showed that a total of 800 credible proteins were identified in both NHA and SHA at 24 and 72 h. In addition, western blot verification showed the expression levels of the tested proteins were found to be generally consistent between the western blot and proteomics results, which demonstrates the reliability of proteomics data in this study.5) Proteomics data were further subjected to the bioinformatics analyses. The results showed that natural and synthetic hydroxyapatites similarly affect GO functional categories, whereas there are some different functions such as "response to inorganic substance", "ion transmembrane transport", "biomineral formation" and "blood vessel development" in NHA and SHA groups. In addition, NHA and SHA could have induced changes in pathways mediating proliferation, adhesion and differentiation. Also, the biological pathways could able to influence each other through protein interaction network.6) The mineralization of cells cultured on disc-shaped NHA and SHA samples was evaluated. The results showed that the degree of mineralization in NHA and SHA was slightly higher than that of the control group. However, NHA exhibited a higher capacity for bone mineralization than SHA.The second part is an integrated analysis of natural hydroxyapatite-induced osteogenic differentiation of mesenchymal stem cells using transcriptomics, proteomics and microRNA analyses. The main contents are as follows.1) It was compared that the proteomics and gene expression profiling data of MSCs cultured on NHA. The results showed that the numbers of up-regulated genes or proteins are greater than the number of down-regulated in the two groups at each time point. Through the GO categories analysis for proteomics data,4 functional nodes associated with osteogenic differentiation were filtered, including skeletal development, regulation of cell differentiation, negative regulation of cell differentiation and positive regulation of cell differentiation. The 4 functional nodes were included in the 13 functional nodes associated with osteogenic differentiation in gene expression profiling data.10 proteins related to NHA osteogenic differentiation was found involved in these 4 functional nodes.2) By integrating transcriptomics and proteomics, the mRNAs and proteins involved in 89 pathways were shown to be differentially expressed. Among them, some pathways such as MAPK and TGF-beta Receptor pathway might play an important role in NHA-induced osteogenic differentiation.3) The regulatory functions of miRNAs in natural hydroxyapatite-induced osteogenic differentiation were analyzed. Among detected 13 miRNAs,9 miRNAs were participated in regulating differentiation-related biological pathways. In which, miR-26a and miR-26b might inhibit adipogenesis through regulating adipogenesis pathway; miR-222 might play an important role in promoting osteogenic differentiation through regulating MAPK signaling pathway.4) The osteoinduction potential of natural hydroxyapatite was evaluated. The ALP staining showed that MSCs cultured on NHA could be induced to osteogenic differentiation. A verification experiment for the osteogenic pathway indicated that both the ERK1/2 and JNK pathways were responsible for NHA-induced osteogenic differentiation, but the ERK1/2 pathway might be predominant.更多还原