【作者】 侯洋；

【导师】 袁文；

【作者基本信息】 第二军医大学 ， 外科学， 2013， 博士

【摘要】 一、研究背景细胞-基质的相互作用对于细胞骨架结构、生长和分化的调节具有重要的意义。细胞的存活依赖于细胞和细胞外基质（extracellular matrix，ECM）、其它细胞以及血清中的生长因子之间的相互作用。如果这些相互作用被阻遏，正常细胞会经历凋亡，这是一种细胞程序性死亡的生理过程。细胞凋亡在维持细胞数目和组织结构方面起着至关重要的作用，其调控机制十分复杂。其中某些细胞-基质的作用是通过整合素蛋白进行调节。这是一种细胞表面受体家族。大多数整合素蛋白通过基质蛋白调节细胞的贴附，这些基质蛋白包括胶原、纤连蛋白和玻连蛋白。整合素可以产生细胞外信号来调节细胞的贴附、形态、分化、增殖和凋亡。整合素可以和数种细胞基质外分子相互作用，包括纤维连接蛋白，骨桥蛋白，骨唾液蛋白，I型， II型和X型胶原。由于这些蛋白包含着特定的氨基酸序列，多种类型的整联蛋白α和β亚基形成非共价的异二聚体以适应每个不同的结合位点来调控细胞应答。P15蛋白（序列GTPGPQGIAGQRGVV）是I型胶原α1链中15个连续的氨基酸。在本研究中，我们用P15蛋白共价结合钛合金表面，并研究P15蛋白是否能够增强成骨细胞的贴附，增殖和分化。此外，我们还探索了整联蛋白的激活是否也参与其中，从而评价P15蛋白作为一种有效的生物活性分子应用于骨组织再生工程可行性。骨形成是细胞和基质进行三维组合的复杂过程。正常骨的形成包括以下两种机制：膜内成骨和软骨内成骨。膜内成骨主要是通过内在的骨膜成骨板成骨，并且不经过软骨形成的阶段。软骨内成骨是在骨骺和骺板软骨形成并且延长以成骨之后，在矿化的软骨支架上进行骨合成，其机制依赖于修复中应力环境。这些机制同样在骨折和截骨后修复发挥作用。通过膜内成骨，间充质干细胞从成骨前体细胞分化为成骨细胞，而软骨内成骨的特征是软骨的合成在先，而之后骨形成是按照软骨内成骨的顺序进行的。本研究探索了外源添加的P15蛋白对于软骨细胞形态，碱性磷酸酶水平，软骨基因的改变以及蛋白表达的作用来研究软骨内成骨是否参与了P15蛋白促进骨形成的作用机制。自体骨移植被认为是骨移植材料中的“金标准”，能够增强骨再生和修复骨缺损，但是取自体骨会带来相关的供体区并发症，进而限制了这项技术的应用。此外，应用异体骨同样也存在不足之处，包括高成本，消毒和存储的问题，并且最重要的是潜在的宿主对外来组织的免疫源性反应和传染病输入的风险。总体上，骨移植材料的需求最近几年一直在增加，原因主要是因为需要骨移植的手术数量增加了，例如脊柱融合手术，关节置换翻修，继发于创伤的保肢手术，肿瘤或者骨畸形。未来的研究主要是克服前述提到的自体骨和异体骨移植物的缺点，进而发展出新的可以作为传统骨移植物的替代品或辅助品的“原生物”材料，从而满足临床治疗对于骨再生的要求。无机牛源性羟基磷灰石（Anorganic bovine-derived hydroxyapatite, ABM)是一种天然的微孔羟基磷灰石复合物（HA），ABM已经用于修补牙周缺损。HA具有很好地生物相容性。其多孔的结构可以用于制作支架和促进骨的生长。然而，HA具有惰性并且骨传导性较差。为了增强骨的生长和诱导新骨的形成，我们将P15蛋白和HA相结合。P15蛋白主要参与成纤维细胞和成骨细胞的贴附。P15蛋白被证明可以促进细胞间贴附，并且可以支持细胞在HA表面的贴附。在本研究中，我们通过小鼠皮下模型和山羊脊柱缺损模型分析评价了ABM/P15作为骨移植替代物的有效性和安全性。二、研究目的（一）探索外源添加的P15蛋白对于软骨细胞的形态、碱性磷酸酶水平以及软骨基因的改变和蛋白表达水平的影响。（二）研究P15蛋白对于特定整联蛋白的激活作用。（三）通过体外小鼠皮下软骨内成骨模型评价P15蛋白对于MSC诱导，软骨细胞分化、和之后的骨化和矿化作用的影响。（四）探索P15蛋白共价结合的钛合金表面对于成骨细胞和间充质干细胞的贴附、增殖和成熟的影响。（五）P15/ABM复合物骨修复效果的评价及P15蛋白的细胞毒性分析。三、内容和方法（一）成软骨细胞微团培养和染色用于观察P15蛋白对于细胞生长分化的影响。（二）双免疫荧光法检测P15蛋白和α5整联蛋白共表达。蛋白免疫印迹实验和免疫组织化学染色检测含有P15蛋白培养基培养的鼠间充质干细胞软骨分化基因的表达。（三）将含有P15蛋白的生物基质胶注射于小鼠皮下，之后通过微型CT扫描、苏木精伊红/阿辛蓝染色和免疫组织化学染色分析P15蛋白对于骨形成作用的影响。（四）乳酸脱氢酶和MTT实验检测P15蛋白共价连接的钛合金表面的细胞毒性。实时定量qPCR、蛋白免疫印迹实验和免疫组织化学荧光检测被用于评价培养细胞的成骨基因表达和分化。（五） P15蛋白在骨形成中的作用通过山羊脊柱缺损模型进行评价。此外，蛋白免疫印迹实验和免疫组织化学荧光检测被用于评价P15蛋白对于人关节软骨细胞和成纤维细胞成软骨分化的影响。LDH和MTT实验被用于研究P15蛋白的细胞毒性。四、研究结果（一）P15蛋白组细胞增殖、生长和分化较对照组明显加快。（二）P15蛋白和α5整联蛋白在P15蛋白组细胞中共表达，P15蛋白组的软骨分化基因表达显著高于对照组。（三）小鼠皮下软骨骨化模型表明P15蛋白组的骨形成体积显著高于对照组，有更多的软骨生成，并且成软骨基因表达高于对照组。（四）接种于P15蛋白共价连接的钛合金表面的细胞LDH和MTT结果与对照组无显著统计学差异（P＞0.05），且可以增强培养细胞的成骨基因表达水平。（五）P15/ABM组山羊脊柱缺损区可以观察到大量的新骨形成，并且未观察到炎性反应的发生。P15蛋白组的成纤维细胞LDH和MTT实验结果与对照组无统计学差异（P＞0.05），且细胞凋亡基因表达水平与对照组无统计学差异（P＞0.05）。五、实验结论（一）P15蛋白可以促进MSC细胞的增殖、生长和成软骨分化。（二）P15蛋白可以通过软骨内成骨促进骨的生长。（三）P15蛋白共价连接的钛金属表面对于MSC细胞无毒性，并且可以使细胞成骨分化的基因表达升高。（四）P15/ABM能够促进脊柱骨缺损的修复，并且不会产生免疫应答。P15蛋白对于成纤维细胞无毒性并且不会诱导细胞凋亡。更多还原

【Abstract】 Back groundCell–matrix interactions are crucial for the regulation of cytoskeletal structure, growthand differentiation. Cell survival depends on cellular interaction with extracellular matrix(ECM), with other cells and with soluble growth factors in the serum. If these interactionsare prevented, normal cells may undergo apoptosis, a physiological form of programmedcell death. Apoptosis provides a vital mechanism for maintaining cell number and tissuestructure with complex mechanisms of regulation. Certain cell–ECM interactions aremediated by integrins, a major family of cell surface receptors. Most integrins mediate celladhesion to matrix proteins, including collagen, fibronectin, and vitronectin. Integrins cangenerate intracellular signals that regulate cell adhesion, morphology, differentiation,proliferation and apoptosis.Integrins can interact with several ECM molecules including; fibronectin, osteopontin,bone sialoprotien, collagen I, II and X. As each of these proteins contain specific aminoacid sequences, multiple types of integrin alpha and beta subunits exist and formnoncovalent heterodimers to conform to each different binding site thereby modulatingcellular response. P15is the common name of a synthetic15amino acid peptide(GTPGPQGIAGQRGVV) whose sequence is taken from the cell-binding domain ofhuman collagen type I protein. In this study, we explore covalently attaching the P15peptide to a titanium surface to evaluate whether it will enhance osteoblastic adhesion,proliferation and differentiation. Additionally, we determine if integrin activation isinvolved in this process to evaluate the feasibility of P15peptide as an effectively bioactivemolecule in the use of bone tissue regeneration.Bone formation is complex but the three-dimensional positioning of cells andmatrices is straightforward. Normal bone develops using only2mechanisms:Intramembranous bone formation and endochondral bone formation. Intramembranousbone formation is mediated by the inner periosteal osteogenic layer with bone synthesizedinitially without the mediation of a cartilage phase. Endochondral bone formation describesthe synthesis of bone on a mineralized cartilage scaffold after epiphyseal and physealcartilage have shaped and elongated the developing organ. These mechanisms are alsoused in fracture and osteotomy repair with the specific mechanism dependent on the mechanical environment provided during repair. With intramembranous bone repair,mesenchymal cells differentiate along a preosteoblast to osteoblast line while endochondralbone repair is characterized by the initial synthesis of cartilage followed by theendochondral sequence of bone formation. This study investigate the effect ofexogenously added P-15peptide on chondrocyte morphology, alkaline phosphatase levels,and alteration of chondrocytic gene and protein expression to analyze whether theendochondral bone formation is involed in the mechanism of P15peptide in the promotionof bone formation.The autologous bone graft is considered to be the “gold standard” bone graftingmaterial to enhance bone regeneration and repair bone defects, its harvesting is associatedwith donor-site morbidity and restricted availability. Furthermore, the alternative use ofallografts has also certain drawbacks, including high cost, issues of processing, sterilizationand storage, and most importantly potential immunogenic response by the host to theforeign tissue and disease transmission. Overall, the requirements for bone graftingmaterial have increased significantly within the recent years, mainly due to the increasingnumber of procedures requiring bone augmentation, such as spinal fusion procedures,revision arthroplasties and limb salvage procedures secondary to trauma, tumour orskeletal abnormalities. Ongoing research to overcome the aforementioned limitations ofautologous and allogeneic bone grafts led to development of new “orthobiologic” materialseither as adjuncts or as alternatives to the “traditional” grafting materials for themanagement of any clinical condition-requiring enhancement of bone regeneration.Anorganic bovine-derived hydroxyapatite （ABM） is a natural microporous HAcompound that is approved for the filling of periodontal defects. HA has excellentbiocompatibility and its porous structure serves as a scaffold and allows bone ingrowth.However, HA is rather inert and has a weak osteoinductive activity. In order to enhancebone ingrowth and to induce new bone formation, we added the cell-binding peptide (P-15)to HA. P-15is a synthetic clone of15-amino acid sequence of type I collagen, which ismainly involved in the adhesion/junction of cells, primarily fibroblasts and osteoblasts.P-15has been shown to have the ability to support the intracellular adhesion; moreover itsupports the adhesion of cells on HA-surface. In this study, we analyze and evaluate theefficacy and safety of the ABM/P15as the bone graft substitute in the mouse subdermalmodel and sheep vertebral defect model. Objective1. To examine the effect of exogenously added P-15peptide on chondrocyte morphology,alkaline phosphatase levels, and alteration of chondrocytic gene and protein expression.2. To investigate the role of P-15peptide on activation of specific integrins.3. An in vivo murine subdermal endochondral ossification model was utilized to evaluateeffect of P-15peptide on MSC attraction, chondrocytic differentiation, and subsequentossification and mineralization.4. To investigated osteoblast and mesenchymal cell (MSC) adhesion, proliferation andmaturation on covalently attached P15-titanium (Ti-P15) surfaces.5. The evaluation of the efficacy of the P15/ABM in the bone repair and the cellularcytotoxicity of P-15peptide.Methods1. The chondrogenic micromass culture and staining were used to observe the effects ofP15peptide on the cellular growth and differentiation.2. The double immunofluorescence was used to test the coexpression of P15peptide andα5integrin. The western blot analysis and immunohistochemistry were performed to testthe expression of chondrogenic genes in mouse stem cells cultured with P15peptide.3. The biological matrigel containing P15peptide was injected subcutaneously to themouse. Then, the microCT, HE and alcian blue and immunohistochemical staining wereused to analyze the effects of P15peptide on bone formation.4. P15peptide was covalently bonded to titanium alloy surfaces. LDH and MTT assaywere used to evaluate the cellular cytotoxity of titanium alloy surface covalently bondedwith P15peptide. The real-time qPCR, western blot and fluorescent immunohistochemistrywere performed to measure osteoblast gene expression and differentiation of the culturedcells.5. The role of the P-15peptide in bone formation was investigated using in vivo sheepvertebral defect and murine subdermal endochondral ossification models. In addition,western blot and fluorescent immunohistochemistry were performed to evaluate the effects of P-15peptide on the human articular chondrocytes and fibroblasts. LDH and MTT assaywere also used to investigate the cellular cytotoxicity of P-15peptide.Results1. The proliferation, growth and differentiation of the cells in P15group are significantlyenhanced compared with control group.2. The coexpression of the P15peptide and α5integrin was observed in P15group. Theexpression of chondrogenic genes in P15group was significantly higher than the controlgroup.3. The mouse subdermal endochondral ossification model showed that the volume of thenewly formed bone in P15group was significantly higher than the control group. The P15group has more newly formed cartilage and higher expression of the chondrogenic genescompared with control group.4. The LDH and MTT results showed no statistical difference between the cells growed onthe P15-titanium surface and those on the titanium surface (P＞0.05), and the P15peptidecan enhance the expression of the osteogenic genes in cells grown on the P15-titaniumsurface.5. Large amount of newly formed bone was observed in the defect area of the sheep spinein P15/ABM group without the presence of inflammatory response. The LDH and MTTtests showed no statistical difference between the fibroblasts in P15group and those incontrol group (P＞0.05). No statistical difference of the expression of apoptosis genes wasfound between the P15and control group (P＞0.05).Conclusion1. P15peptide can enhance the proliferation, growth and chondrogenic differentiation ofMSCs.2. P15peptide can promote the bone growth by the endochondral bone formation.3. The P15-titanium has no cellular cytotoxicity to the MSCs and can elevate theexpression of osteogenic genes of MSCs. 4. P15/ABM can facilitate the bone repair in the spinal defect area without theinflammatory response. P15peptide has no cellular cytotoxicity to the fibroblasts andcannot induce the apoptosis.更多还原