【作者】 范志海；

【导师】 董启榕； 沈忆新；

【作者基本信息】 苏州大学 ， 骨外科学， 2014， 博士

【摘要】 脊髓损伤（spinal cord injury，SCI）不可避免造成神经元死亡及其轴突断裂，进而导致神经支配功能的丧失。支持和引导轴突生长是SCI修复的关键。作为支持和引导损伤神经元轴突生长的移植细胞，嗅鞘细胞（olfactory ensheathing cells，OECs）具有天然的优势。然而，SCI后胶质细胞表达的抑制分子形成抑制轴突再生的化学屏障，胶质细胞活化迁移在损伤部位形成的胶质瘢痕和空洞缺损成为轴突再生的物理屏障。在此环境中，移植细胞难以定向迁移和有序分布，无法为再生轴突提供有效的支持和引导，这是细胞移植修复SCI难以取得突破的重要原因。因此，通过组织工程构建仿生支架定向分布移植细胞，克服损伤部位抑制分子和瘢痕空洞对轴突再生的不利影响，为神经元轴突再生提供支持和引导，成为实现SCI功能修复的重要突破口，也是目前SCI修复的研究热点。选择性能优异的生物材料是研究的前提。丝素蛋白（silk fibroin, SF）以其优异的生物相容性、良好的力学性能、可控的降解和可塑性能逐渐被研究者认知。支架仿生构建关键在于对基质材料纳米结构的调控。近年来，SF的自组装机制和纳米结构调控研究取得重大进展。基于SF自组装机制的研究，本项目组获得结构均一的纳米线结构单元，发现该纳米线结构对SF静电纺丝过程的纤维直径及走向具有重要影响，可以使得SF纳米纤维的直径更为可控、取向更为有序。因此，作为组织工程支架的基质材料，SF具有天然的优势。为探讨SF纳米纤维材料作为定向分布OECs的移植载体的可行性，本课题就以下几方面内容进行探讨：（1）建立OEC培养体系，倒置相差显微镜下观察培养周期内细胞形态的变化，活细胞工作站观察OECs连续动态的形态变化，明确同一培养条件下OECs迁移运动的形态特征及其变化规律。（2）研究不同培养条件（有或无血清，不同增殖剂：Forskolin或bFGF）对OECs的形态及其迁移能力的影响，明确不同培养条件下OECs迁移运动的形态特征及其变化规律，分析OECs迁移的调控机制。（3）静电纺丝工艺制备桑蚕丝（BSF）和柞蚕丝（TSF）纳米纤维支架，检测比较并评价两种支架材料对OECs的形态、增殖、存活及其表型的影响，明确SF纳米纤维支架的生物相容性及其优越性的具体体现。（4）扫描电镜观察SF支架及细胞的超微结构，光镜下观察细胞的形态及其分布，免疫荧光染色显示细胞表型及细胞骨架的发育，活细胞工作站连续动态观察细胞形态特征，明确其变化规律，分析细胞迁移轨迹、迁移速度、迁移速率等，研究静电纺SF纳米纤维支架的不同种类（TSF与BSF）、不同直径（400nm和1200nm）和不同取向分布（平行有序排列和无序网络状）及空间拓扑结构对OECs迁移运动的影响，明确SF纳米纤维支架定向分布OECs的调控作用及其机制。（5）RT-PCR检测OECs的MBP、NGF、GDNF、BDNF、NCAM、VEGF的基因表达，ELISA检测培养液上清中的蛋白含量，明确SF纳米纤维定向分布的OECs表达功能基因和蛋白量的变化，分析其神经营养功能。（6）探讨SF纳米纤维定向分布OECs对神经元生长发育的影响，明确定向分布OECs的生物学功能，为进一步优化设计及其应用提供理论基础和实验依据。通过上述研究，本课题得出如下结论，并分析其原因、意义及启示：（1）通过活细胞工作站连续动态观察发现，在同一培养条件下，OECs迁移过程中表现出多样性变化的形态特征。分析细胞形态变化与迁移运动之间的关系，明确OECs多样性的形态表现是由其功能状态决定的，不同形态的OECs适应不同的功能状态：细长突起及梭形胞体形态有利于细胞迁移运动，扁平状有利于胞体铺展黏附，圆球状有利于细胞生长增殖。OECs迁移运动的形态特征及其变化规律更加合理地解释OECs的形态多样性。在电镜、光镜和免疫染色观察细胞的形态都是相对静止和固定的，因而难以区分和正确理解细胞形态的多样性是由于细胞适应其自身功能需要而形变的结果。进一步明确OECs形态变化与迁移运动功能之间的相互关系有助于理解OECs迁移的调控机制。（2）通过研究不同培养条件对OECs的形态及其迁移能力的影响，明确OECs具有优异的可塑性。OECs自胞体伸出微小突起感知周围环境信号变化，从而改变自身形态以适应环境变化，进而实现细胞不同功能的表达。无血清条件培养液、Forskolin和bFGF有利于OECs运动功能的表达，提高了细胞的迁移速度。由于OECs迁移缺乏有效引导，迁移轨迹和迁移方向是随机和无规律性的，呈现无规行走。实现信号分子的浓度梯度分布或是构建模拟细胞外基质三维纳米空间结构的仿生支架是体外引导和调控细胞定向迁移的有效途径。（3）SF（尤其是TSF）纳米纤维支架具有良好的生物相容性，能够促进OECs黏附、生长与增殖。分析原因：SF纳米纤维支架高的表面与体积比率、表面活性和优异的亲水性能有利于支架材料吸附培养液中的蛋白质分子和其它生物活性物质，从而促进细胞在支架表面黏附，进而有利于细胞的存活、生长与增殖。TSF纳米纤维具有更为优异的生物相容性，与其含有丰富的精氨酸-甘氨酸-天冬氨酸(RGD)三肽序列有关，RGD序列是人类细胞外基质中广泛存在的能够被细胞表面受体识别和特异性结合的序列，作为细胞膜整合素受体与细胞外配体相结合的识别位点，介导细胞与细胞外基质及细胞之间的相互作用，能够促进细胞对支架的黏附，进而促进细胞的生长与增殖。TSF富含RGD三肽的特性赋予其更为优异的生物学性能。（4）SF纳米纤维的直径、取向分布及空间拓扑结构对定向分布OECs的调控作用。OECs对BSF和TSF纳米纤维的不同直径大小、取向分布及三维空间拓扑结构存在不同的生物学响应。小直径（400纳米）对促进细胞的黏附、生长、增殖及迁移都有更为优异的表现。分析原因：OECs依赖于其突起寻路及附着，其细小的突起更容易黏附于小直径的纳米纤维之上，进而有利于实现细胞的生长、增殖及迁移等生物学功能。平行排列的纳米纤维有利于引导和调控细胞迁移轨迹和方向，细胞沿纳米纤维的取向形成有序分布，在同一根纤维上，细胞间的突起相互连接从而细长串珠样结构。分析原因：平行排列的纳米纤维结构能够使细胞的突起在寻路过程中不易被其它纤维或细胞影响和干扰，因而在迁移过程中能够保持较好的方向性，引导细胞在支架材料上定向迁移并有序分布。从超微结构观察细胞在支架三维拓扑结构中的状态，能够提示支架的孔隙大小、纤维间的距离及排列方式对细胞的形态及功能也有重要影响，这对于构建体内应用的支架有着重要的启示意义。目前SF纳米纤维的直径能够控制在50纳米级别，同时纤维取向分布更为有序，今后将更为深入探讨纤维直径、取向及三维空间拓扑结构对OECs生物学行为的影响，以进一步优化支架的结构设计。（5）SF纳米纤维调控OECs定向迁移和有序分布与细胞骨架的排列方式密切相关。从细胞骨架形态及其分布的观察可以看出，生长在SF纳米纤维上的OECs细胞骨架发育理想，微管蛋白排列整齐，表明细胞可以感受生长环境，对SF纳米纤维支架具有很好的反应，而且对小直径平行排列的TSF纳米纤维的反应更强。同时提示，OECs对基质环境的响应是通过调控细胞骨架的形成及其架构，从而控制细胞的形态变化（如突起的延伸及回缩，胞体的形变），进而促进细胞的迁移并控制细胞迁移的方向，引导细胞在支架材料上形成有序分布。（6）TSF纳米纤维支架有利于OECs功能基因和蛋白的表达。TSF纳米纤维支架为细胞提供的生长环境有利于细胞合成与分泌细胞外基质成分、细胞黏附分子及可溶性趋化因子，使得细胞能够在支架上迁移和相互聚集，细胞间形成更多的通讯联接。同时，TSF纳米纤维支架促进细胞分泌细胞外基质的作用还可以进一步促进细胞的生长与增殖。（7）定向分布OECs支持神经元生长发育，引导神经元轴突延伸。支持和引导神经元及其轴突的生长及延伸是OECs在体内嗅觉神经元生长发育及损伤再生过程中作为细胞底物作用的关键，也是其应用于移植修复SCI的重要理论基础。体外构建SF仿生支架定向分布OECs，支持神经元生长发育，引导神经元轴突延伸是本课题研究的目的，也是其研究价值的核心体现。天然细胞外基质的纤维尺寸为50-500nm，进一步控制纳米纤维的直径及取向，最大程度地仿生体内细胞外基质结构是今后研究的重要内容。基于SF自组装机制研究的突破，具有天然细胞外基质胶原纳米纤维结构的SF纳米纤维水凝胶的成型机理及其制备技术是本课题今后的研究方向。此外，利用SF纳米纤维载药缓释技术负载相关蛋白因子，赋予支架生物活性，是实现SF仿生支架修复SCI取得突破的重要环节。更多还原

【Abstract】 Spinal cord injury (SCI) often leads to permanent paralysis and loss of sensationbelow the site of injury because of the inability of damaged axons to regeneration in theadult central never system. Functional deficits following SCI result from damage to orseverance of axons, loss of neurons and glia, and demyelination. The axon growth is thekey to SCI repair. Olfactory ensheathing cells (OECs) in adults remains the ability toinduce axonal growth, making lifelong regeneration ability of olfactory nerve. Therefore,OECs have become a popular candidate for the transplant-mediated repair of the damagedSCI. However, the inhibited molecules formed after SCI will result in the formation ofchemical barrier which inhibited the axonal regeneration. Additionally, glial scar formedin the injured site due to the immigration of activating glial cell, and the nerve defectsbecome the physical barrier suppress the axonal regeneration. In this negative environment,the directional migration and orderly distribution of transplanted cells is impossible, andthe role of transplanted cells in supporting and guiding the axons regeneration is alsodifficult to play, these problems is the main reason that cell transplantation in SCItreatment is unsuccessful. Thus, the directional distribution of transplanted cells throughtissue engineering bionic scaffolds can overcome the adverse effects of the inhibitedmolecules in injury site, scar tissue, and nerve defects, providing support and guidance fornerve axon regeneration, which is not only the important direction for healing SCI, but alsoa hot topic in the research of SCI repair.The presupposition of tissue engineering approaches for management of SCI is to finda biological material with excellent performance and construct a bionic structure. Silkfibroin (SF), a kind of natural protein produced by silkworm, is well known for itsapplication in biomedical areas due to its excellent properties such good biocompatibility,outstanding mechanical properties, controllable degradation, biodegradability, and versatileprocessability. The key to construct biomimetic structure is the control on the nanostructures of scaffolds. Recently, the self-assembly mechanism and regulation onstructure of SF has been further clarified. Based on these progresses, our group controls SFto assemble into homogeneous nanofibrils which will significantly affect the diameter ofelectrospun SF nanofiber, making the fiber diameter control available. Therefore, SF is apromising candidate as a matrix material for tissue engineering scaffolds.To explore the feasibility of SF scaffold as a carrier to directional distributestransplanted OECs, the project plans to study the following aspects:（1）Establishing OECsculture system, inverted phase contrast microscope is employed to observe morphologicalchanges of cell within its cycle; On this basis, the cell station is used to monitor the OECs’migration dynamically, reveal the regularities of OECs’ behavior under the same cultureconditions.（2）Based on the above recognitions, further study is designed to clarify theeffect of culture conditions (such as with or without serum; different proliferation agents:Forskolin, bFGF) on the morphology and migration behavior of cell, and analyze theregulatory mechanisms on OECs migration.（3）Based on bombyx mori silk fibroin (BSF)and tussah silk fibroin (TSF), we prepare SF nanofiber by using electrospinning technique,and culture OECs on these nanofibrous materials. This experiment is designed to study theeffect of chemical composition of SF on the morphology, proliferation, survive, andphenotype of OECs, and clearly define the superior biological performance of TSF insupporting the growth of OECs.（4）Scanning electron microscope, optical microscope,immunostaining, cell station are employed to research the effect of silk fibroin types (TSFand BSF), fiber diameters (400nm and1200nm), and fiber orientations (parallel anddisordered arrangement) of nanofibrous scaffold on the ultrastructure, morphology anddistribution, cell phenotype and cytoskeleton, cell migration behavior (migratory path,migration speed) of OECs during culture in vitro, clearly define the role of topologicalstructure of silk fibroin scaffolds in regulating cell behavior and its regulation mechanism.（5）We employed RT-PCR to detect the gene expression, such as MBP, NGF, GDNF,BDNF, NCAM, and ELISA to detect related protein expression of OECs cultured on silkfibroin scaffolds. Through the investigation, further reveal the role of scaffold in regulatingcell behavior in a molecule and protein level.（6）To explore the effect of SF nanofibrousscaffold on the directional arrangement of OECs, clarify the biological role of SF scaffoldsin targeted distributing OECs, analyze the technique and its development direction inconstruct bionic scaffold, providing theoretical basis and experimental evidence for further scaffold design and its application. Through the above research the conclusions are asfollows:（1）This study employed cell station to continuous and dynamic observe themorphological changes of OECs during their migration, in this situation, the morphologicaldiversity is the result of OECs’ migration, which is attributed to the different functionalstates. The morphological diversity of OECs cultured in vitro is the result of cell migration,which shows the different function states of OECs. It is difficult to find a reasonableexplain for the morphology difference of OECs observed by light microscope and electronmicroscope, in which cell is observed in a static and fixed state, making it difficult todistinguish and correct understanding of cell morphology diversity. The clearly define therelationship between the morphology of OECs cultured in different conditions and cellmigration behavior is helpful to understanding the regulation mechanism of OECsmigration.（2）The strong deformability, active migration ability, and unique role in nerverepair of OECs are attributed to their morphological plasticity. The morphological changes,the formation of pseudopodia, and the migration ability of OECs are regulated by thesignaling molecules in the culture conditions, suggesting the response of OECs tomicroenvironment is sensitive. However, the microenvironment generated by cultureconditions has limited impact on cell migration rate, migration path, and migrationdirection, indicating that orderly distribution of signaling molecules or construction ofnanostructure mimicking ECM is the effective method to guide and regulate cell migrationunder designed direction.（3） SF (especially TSF) nanofiber scaffolds possess good biocompability.Electrospun BSF and TSF fibers are promising growth matrices for OECs due to highsurface to volume ratio and excellent hydrophilicity. The fiber diameter and orientationhave a significant effect on cell adhesion, proliferation, and arrangement. In addition, TSFfibers show more excellent biological properties might be perpetuated by the presence ofthe Arg-Gly-Asp (RGD) sequence and the hydrophilicity.（4）Regulation of the biological behavior of OECs through fiber diameter,orientation, and topological structure of SF nanofibers. The different cell responses tomicro and nano fibers of SF and TSF demonstrated the significant effect of fiber diameteron cell biological behavior. It is found that the fibrous scaffold with small fiber diameter (400nm) is favourable for cell adhesion, proliferation, growth, migration, and expressionof the gene protein. The aligned nanofiber scaffolds is more conducive to cell migration toform targeted arranged cell morphology. OECs on aligned SF nanofibers showed orderedand parallel arrangement, and particularly slender projections, and formed string-structureon a single nanofiber due to the ecphyma connection between cells. The process of findingmigration direction and way of cell ecphyma is disturbed uneasily by other fiber and cell,which keep the migration direction very well during the migration process, resulting in thetargeted and ordered distribution of OECs. At present, the research team has the ability tocontrol the diameter of SF nanofibers down to50nm, providing a platform for furtherinvestigate the effects of fiber with more smaller diameter on cell behavior, thuscontributing to the repair of damaged nerve functions.（5）The control of OECs’ migration and directional distribution by SF nanofiber isclose related to cell cytoskeleton. The biological responses of OECs to fiber diameter, fiberorientation, and3D topology of BSF and TSF nanofibrous scaffolds are different. OECsgrow very well (tubulin in the correct order) on SF nanofiber according to cellcytoskeletonand its arrangement, indicating the ability of cell to respond environment. Thefibrous scaffolds control cell morphology (such as the extension and retraction of theprotrusions, soma deformation), promote cell migration, induce the migration direction,and then control the arrangement by regulating the formation and structure of the cellcytoskeleton. TSF nanofibers exhibited more strong guidance role in regulating cellmigration behavior.（6）TSF nanofiber promote the expression of functional gene and protein of OECs.TSF nanofibers not only have no negative influence upon the expression and secretion ofthese factors of OECs, but promote some protein expression. The reason as follow: TSFnanofibers provide a environment mimicking ECM structure that is conducive to proteinsynthesis, secretion of extracellular matrix components, cell adhesion molecules andsoluble chemokines, making cell migration and aggregation on scaffolds, and formation ofmore communication like between cells, which is important for tissue regeneration andrepair. Meanwhile, the secretion of extracellular matrix by cells induced by TSF nanofiberscaffold would further promote cell growth and proliferation.（7）Directional distribution of OECs support the growth and development of neuron,guide axon extension of neuron. It is the key for OECs as substrates cell to support and guide the growth of neurons and their axons to promote the growth and development ofolfactory nerve and repair of damaged nerve in vivo. The directional arrangement of OECsby TSF nanofibrous scaffolds constructed in vitro is the core values of this research torepair injured neuron and its axons. The results showed that TSF nanofiber scaffold notonly support and promote OECs adhesion, growth, proliferation and gene expression andprotein secretion, but also guide cell orientation align the nanofibrous direction, theorientation distribution OECs support the growth of neurons, and promote the growth ofneurons and their axons.Natural extracellular matrix composed of collagen fiber with diameter size in therange of50-500nm, thus control the diameter and orientation of nanofibers precisely toconstruct scaffolds with maximum bionic ECM structure is an important part of futureresearch. In addition, based on the research breakthroughs of silk fibroin self-assemblymechanism, it is another important direction to form SF hydrogel with controllable gelbehavior and bionic structure. Silk fibroin nanofibers containing drug or related proteinfactor given the biological activity scaffold, in combination of OECs as transplanted cellwould be one of the feasible strategy to repair SCI, and achieve functional recovery.更多还原