本文選題：羥基磷灰石 + 間充質(zhì)干細(xì)胞��； 參考：《東南大學(xué)》2015年博士論文

【摘要】：目前,骨損傷修復(fù)存在的主要困難之一是可用于填充缺損和促進(jìn)骨生長的生物材料非常有限。羥基磷灰石是構(gòu)成骨骼的主要無機(jī)成分,由于具有良好的生物相容性、骨傳導(dǎo)性和骨誘導(dǎo)性,現(xiàn)已被廣泛應(yīng)用于人體骨組織修復(fù)中。羥基磷灰石的主要制備方法包括化學(xué)合成(稱之為合成羥基磷灰石)和從骨骼和牙齒、魚鱗等天然生物體內(nèi)提取(稱之為天然羥基磷灰石)兩種方式。合成羥基磷灰石是一個(gè)化學(xué)分子式為Ca10(PO4)6(OH)2的化學(xué)計(jì)量材料；而天然羥基磷灰石除了羥基磷灰石的成分以外還包含與骨無機(jī)成分相似的微量離子,因此具有與骨礦化成分最大的相似性。目前,基于天然與合成羥基磷灰石醫(yī)用生物材料均已被廣泛使用,但對于天然與合成羥基磷灰石的理化性質(zhì)的對比研究仍然較少,對于兩種材料生物相容性和堿性磷酸酶活性等成骨性能的比較更少,更未見采用蛋白質(zhì)組學(xué)等高通量技術(shù)比較和研究其成骨誘導(dǎo)機(jī)理的報(bào)道。本課題組前期對天然與合成羥基磷灰石進(jìn)行了制備與表征,然后對天然與合成羥基磷灰石的蛋白吸附特性進(jìn)行了比較,并進(jìn)一步通過基因表達(dá)譜芯片技術(shù)結(jié)合生物信息學(xué)分析研究了天然羥基磷灰石對小鼠骨髓間充質(zhì)干細(xì)胞成骨分化的影響。在前期研究的基礎(chǔ)上,本論文將對天然與合成羥基磷灰石的理化性質(zhì)、生物學(xué)性能、骨修復(fù)能力及其分子機(jī)理進(jìn)行系統(tǒng)的比較,然后通過對天然羥基磷灰石作用于細(xì)胞后的轉(zhuǎn)錄組學(xué)、蛋白組學(xué)和miRNA的聯(lián)合分析,探討天然羥基磷灰石誘導(dǎo)骨髓間充質(zhì)干細(xì)胞成骨分化所介導(dǎo)的生物學(xué)通路及通路中miRNA的調(diào)節(jié)作用。本論文包括兩個(gè)部分的內(nèi)容,第一部分是天然和合成羥基磷灰石理化性質(zhì)及生物學(xué)性能比較研究,主要內(nèi)容包括：1)對天然與合成羥基磷灰石粉體和圓盤狀試樣進(jìn)行了制備與理化性質(zhì)的表征,結(jié)果顯示,與合成HA相比,天然HA中存在特有的碳酸根、磷酸氫根及鎂和鈉元素；并且天然羥基磷灰石具有更大的單晶尺度和更高的結(jié)晶度;此外,浸提液中鈣離子的檢測表明天然HA可能具有更高的穩(wěn)定性,而合成HA則具有更高的溶解-沉積特性；鎂離子檢測表明僅在天然HA組出現(xiàn)鎂離子溶出。2)采用貼壁篩選與密度梯度離心分離相結(jié)合的方法對小鼠骨髓間充質(zhì)干細(xì)胞進(jìn)行了分離提取,然后采用流式細(xì)胞術(shù)進(jìn)行鑒定了細(xì)胞表面標(biāo)志物和細(xì)胞的同質(zhì)性,結(jié)果顯示,第4代間充質(zhì)干細(xì)胞高表達(dá)細(xì)胞粘附因子CD29和CD44,而低表達(dá)淋巴細(xì)胞標(biāo)志物CD14和造血細(xì)胞標(biāo)志物CD34,這表明第4代細(xì)胞已經(jīng)得到了純化,所培養(yǎng)的細(xì)胞為骨髓間充質(zhì)干細(xì)胞。3)采用熒光染色和MTT測定方法研究間充質(zhì)干細(xì)胞在天然與合成羥基磷灰石圓盤狀試樣表面的形貌和增殖情況,結(jié)果顯示,天然與合成羥基磷灰石表面所培養(yǎng)的細(xì)胞形貌相似,與對照組相比材料表面細(xì)胞更易呈團(tuán)聚狀生長且增殖緩慢。4)采用基于iTRAQ標(biāo)記的二維液相色譜串聯(lián)質(zhì)譜聯(lián)用技術(shù)對天然與合成羥基磷灰石表面生長細(xì)胞的蛋白質(zhì)表達(dá)情況進(jìn)行研究,在天然與合成羥基磷灰石組24和72小時(shí)共鑒定出可信蛋白質(zhì)800個(gè)；對所選蛋白質(zhì)的表達(dá)情況進(jìn)行Western blot驗(yàn)證顯示兩種方法所得結(jié)果具有較好的一致性,這表明蛋白質(zhì)組學(xué)數(shù)據(jù)可信。5)對蛋白質(zhì)數(shù)據(jù)進(jìn)行生物信息學(xué)分析,結(jié)果顯示天然與合成羥基磷灰石對細(xì)胞主要的GO功能類別影響相似,但在“對無機(jī)物質(zhì)的反應(yīng)”、“離子跨膜運(yùn)輸”、“生物礦化”和“血管發(fā)育”等功能中存在一定的差異；此外,兩種材料能夠通過特定生物學(xué)通路介導(dǎo)細(xì)胞的增殖、形貌和分化功能,并且生物學(xué)通路之間能夠通過蛋白質(zhì)相互作用網(wǎng)絡(luò)而相互聯(lián)系。6)對培養(yǎng)于天然與合成羥基磷灰石表面的細(xì)胞進(jìn)行礦化檢測,結(jié)果表明兩種材料表面細(xì)胞的礦化程度均高于對照組,但天然羥基磷灰石能夠?qū)ΦV化作用產(chǎn)生更大的促進(jìn)作用。第二部分是天然羥基磷灰石成骨誘導(dǎo)機(jī)理的轉(zhuǎn)錄組學(xué)、蛋白質(zhì)組學(xué)和miRNA聯(lián)合分析,主要內(nèi)容包括：1)對天然羥基磷灰石對細(xì)胞作用的蛋白質(zhì)組學(xué)和基因表達(dá)譜芯片數(shù)據(jù)進(jìn)行比較,結(jié)果顯示,兩組數(shù)據(jù)在各時(shí)間點(diǎn)的上調(diào)表達(dá)基因或蛋白質(zhì)數(shù)量均大于下調(diào)表達(dá)的基因或蛋白質(zhì)數(shù)量；對蛋白質(zhì)組學(xué)數(shù)據(jù)進(jìn)行GO功能分析共篩選到4個(gè)與成骨分化相關(guān)的節(jié)點(diǎn),分別為骨骼發(fā)育、細(xì)胞分化調(diào)控、細(xì)胞分化負(fù)向調(diào)控和細(xì)胞分化正向調(diào)控。這4個(gè)節(jié)點(diǎn)包含在基因表達(dá)譜芯片數(shù)據(jù)所涉及的13個(gè)與成骨分化相關(guān)的節(jié)點(diǎn)中。在這4個(gè)節(jié)點(diǎn)中最終篩選得到10個(gè)與成骨分化相關(guān)的蛋白質(zhì)。2)對蛋白質(zhì)組學(xué)數(shù)據(jù)與基因表達(dá)譜芯片數(shù)據(jù)進(jìn)行生物學(xué)通路聯(lián)合分析,共得到89條嬖RNA和蛋白質(zhì)共同參與的生物學(xué)通路,其中MAPK信號(hào)通路和TGF-β受體信號(hào)通路等可能在天然羥基磷灰石所誘導(dǎo)的成骨分化中發(fā)揮重要作用。3)對microRNA在天然羥基磷灰石成骨誘導(dǎo)中的調(diào)節(jié)作用進(jìn)行分析,結(jié)果顯示在所檢測的13個(gè)microRNA中,有9個(gè)microRNA在分化相關(guān)通路中發(fā)揮了調(diào)控作用,其中miR-26a和miR-26b可能通過調(diào)控成脂分化通路從而對細(xì)胞的成脂分化產(chǎn)生抑制作用,而miR-222可能通過調(diào)控MAPK通路從而在細(xì)胞的成骨分化中發(fā)揮重要的促進(jìn)作用。4)對天然羥基磷灰石的成骨誘導(dǎo)能力進(jìn)行了檢測,堿性磷酸酶染色實(shí)驗(yàn)顯示培養(yǎng)于天然羥基磷灰石表面的間充質(zhì)干細(xì)胞能夠被誘導(dǎo)成骨分化；成骨分化通路驗(yàn)證試驗(yàn)表明,天然羥基磷灰石通過MEK1/2-ERK1/2和JNK MAPK通路共同介導(dǎo)細(xì)胞的成骨分化,其中MEK1/2-ERK1/2 MAPK通路在成骨誘導(dǎo)中發(fā)揮主要作用。
[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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R68;R318.08
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本文編號(hào)：2109530