大腹园蛛管状腺丝重组蛋白的克隆表达及纺丝

TuSp1蛋白(tubuliform spidroin 1)是管状腺丝(tubuliform silkfiber)的主要组成成分。管状腺丝作为蛛丝卵袋的外层包卵丝,其结构具有很好的耐腐蚀性和良好的力学性能。目前国内外对大腹园蛛TuSp1蛋白的研究很少,仅有一条基因序列的报道。本课题首次构建含大腹园蛛N端非重复结构域、重复单元以及C端非重复结构域的重组管状腺丝蛋白TuSp1 NT-Rp-CT,并经湿法纺丝获得重组蛋白丝纤维。重组蛋白液圆二色谱分析结果显示,pH由7.0降低到5.5的过程中,始终保持稳定的α-螺旋构象;重组蛋白丝纤维的傅里叶变换红外光谱结果显示,丝纤维中主要二级结构为β-折叠及β-转角;经扫描电镜观察发现,冻干的絮状重组蛋白能自组装成丝纤维,且表面光滑纤细;湿纺后的重组蛋白丝纤维直径较粗,但表面较平整均匀,具有类似天然管状腺丝的形态特征,这些为TuSp1蛋白的成丝机理及仿生纺丝研究提供了理论依据。     

牛脊髓神经丝的体外组装与结构

利用差速离心法从牛脊髓中分离神经丝 ,在电镜下观察了其形态 ;应用扫描隧道显微镜 (STM)研究了它的结构 ,发现神经丝具有长短 2种侧臂 ,二者相间排列 ,相邻长侧臂或相邻短侧臂的间距都是 2 0～ 2 2nm ;由此推测神经丝内部存在 3 /4分子交错 ;还研究了神经丝蛋白的体外组装 ,以胶体金标记的方法证明 ,中等分子量与高分子量的神经丝蛋白 ,都能同低分子量的神经丝蛋白共同装配成 10nm的纤维 ;同时发现 ,中等分子量与高分子量的神经丝蛋白能够组装成一种较细的纤维 ,不同于中间纤维 .     

新型细胞骨架分隔丝的结构和功能研究进展

细胞骨架是细胞内的蛋白纤维网状结构,包括人们熟知的微管、微丝和中间纤维.目前研究表明分隔丝(septin filaments)是一类在真核生物中广泛分布的蛋白纤维,逐渐被认为是一种新型细胞骨架结构.分隔丝由可结合GTP的分隔丝蛋白单体(Septin)聚合形成异源复合体,进一步组装成纤维丝.分隔丝可形成纤维束,环状或笼状等结构,并与细胞膜或其他细胞骨架成分发生相互作用.在细胞内,分隔丝参与胞质分裂、细胞迁移、神经元发育和免疫等重要生理及病理过程.分隔丝结构或功能的异常与多种人类疾病如肿瘤等密切相关.本文将从分隔丝的结构、组装调控、功能及与人类疾病的关系等方面综述近年的研究进展.     

丝心蛋白的研究及在医药等方面的应用

丝心蛋白的研究及在医药等方面的应用吉爱国汲萍（山东大学药学系,济南２５００１２）（山东医科大学口腔系）关键词丝心蛋白丝心蛋白俗称丝素,是以脱胶天然蚕丝为原料制成的粉状纤维蛋白质。近年来,随着生物技术和其它高新技术的迅速发展和广泛应用,国内外对丝心蛋白...     

血管新生及丝蛋白材料血管化过程

基于医用生物材料开发及组织工程中血管化问题的重要性,本文就与生物材料血管化紧密相关的血管发生和血管新生有关研究做一综述,分析了芽式和套迭式血管新生的模式及机制,特别是对丝蛋白材料的血管化过程进行了分析与探讨.通过深入探讨血管新生的模式和机制,进而阐明丝蛋白材料中毛细血管生长与生物材料微结构之间的关系,有助于设计出适合于细胞黏附、组织生长、血管化顺利进行的生物材料,促进生物材料的临床应用及组织工程血管化研究的深化.     

蛋白质内含子介导蛛丝功能化平台的建立

为建立高效快捷的蛛丝功能化修饰平台,蛋白质内含子的反式剪接技术被首次应用于重组蛛丝的功能化修饰。在体外通过Ssp Dna B的反式剪接作用,在蛋白质水平上将12 k Da泛素相关修饰蛋白(SUMO)与蛛丝蛋白(W2CT)连接形成功能化蛛丝蛋白SUMOW2CT。修饰后SUMOW2CT与W2CT均能形成纳米至微米级的丝纤维,但SUMOW2CT自动成丝速度明显下降且产量约为W2CT的一半。与W2CT丝纤维(W)相似,SUMOW2CT丝纤维(UW)不具有超收缩能力和对2%SDS不耐受,但机械性能低于W2CT丝纤维。功能化蛋白SUMOW2CT形成的丝纤维中SUMO蛋白仍保持着正确三维结构,可被SUMO蛋白酶酶切。外源功能化蛋白质虽在一定程度上降低了丝的形成速度和机械性能,但修饰上的功能化蛋白仍保持着生物活性,表明断裂蛋白质内含子介导的蛛丝修饰平台成功建立,也为蛛丝的功能化修饰和应用奠定了坚实的技术基础。     

蚕丝纳米纤维在组织工程领域的研究进展

在组织工程中最为关键的是提供理想的生物材料支架,而结构和功能仿生是组织工程支架最重要的性能要求,但仿生组织工程支架的设计与构建必须由纳米纤维来实现.本文介绍蚕丝纳米纤维的性能特点,并通过国内外文献证实了家蚕丝素具有良好的生物相容性等优良特性,在组织工程领域广泛应用.然而,与家蚕丝相比,野蚕丝更利于细胞的黏附、生长,因此我们推测利用我国特有的野蚕丝素进行静电纺丝制取纳米纤维,有望在生物医学领域获得新的进展.     

蜘蛛丝蛋白研究进展

由于蜘蛛丝蛋白分子高度重复的一级结构、特殊的溶解特性和分子折叠行为以及具有形成非凡力学特性丝纤维的能力而引人注目。本文从蛛丝蛋白基因、天然蛛丝形成过程、蛛丝蛋白的基因工程生产及蛛丝蛋白的应用前景等几个方面着重介绍了近20年来对蛛丝蛋白的研究进展。围绕蛛丝蛋白展开的研究将有助于揭示蛋白质一级结构、蛋白质分子折叠与蛋白质大分子特性之间的内在联系。     

低分子量神经丝蛋白(NF-L)的体外组装

利用超速离心和离子交换层析技术,从牛脊髓中分离纯化了神经丝蛋白三组分:NF-L,NF-M和NF-H。应用电镜负染色和金属投影方法研究神经丝的形态结构与NF-L的体外组装,结果表明:神经丝由10nm的核心纤维与外周的丝状突起组成;在近似生理条件下,NF-L可在60min内组装成10nm纤维,纤维由4根亚丝缠绕而成;在碱性缓冲液中,NaCl能促进NF-L装配成短纤维,这种10nm的短纤维无法连接成长纤维。     

次壶腹腺丝蛋白功能模块的研究

蜘蛛丝是天然的生物材料,具有潜在的巨大应用价值。研究蜘蛛丝蛋白质的结构与功能,有助于破解蜘蛛丝蛋白质的成丝机理,为制备优良材料学性能的仿生蜘蛛丝纤维提供理论依据。以MiSp蜘蛛丝的重复区和C端非重复区蛋白多肽为研究对象,在不同pH值和离子条件下,在体外研究其二级结构与成丝的关系。CD图谱显示:表达纯化的重组蜘蛛丝蛋白R1R2在pH 7.5、6.5和5.5时二级结构相似,均为无规则卷曲,而R1R2CT则主要呈现为α螺旋构象;扫描电镜结果表明:在以上3种pH条件下,只有pH 5.5时R1R2和R1R2CT才形成重组丝纤维,R1R2CT纤维形态较平整,类似于天然蛛丝纤维形态,而R1R2丝纤维则呈条带状,表面粗糙。另外,氯化钠不利于形成形态平整的丝纤维。该成果为研究蛛丝蛋白的成丝机理奠定基础,也为制备仿生蛛丝蛋白纤维提供理论依据。     

Use of spider silk fibres as an innovative material in a biocompatible artificial nerve conduit

Defects of peripheral nerves still represent a challenge for surgical nerve reconstruction. Recent studies concentrated on replacement by artificial nerve conduits from different synthetic or biological materials. In our study, we describe for the first time the use of spider silk fibres as a new material in nerve tissue engineering. Schwann cells (SC) were cultivated on spider silk fibres. Cells adhered quickly on the fibres compared to polydioxanone monofilaments (PDS). SC survival and proliferation was normal in Live/Dead assays. The silk fibres were ensheathed completely with cells. We developed composite nerve grafts of acellularized veins, spider silk fibres and SC diluted in matrigel. These artificial nerve grafts could be cultivated in vitro for one week. Histological analysis showed that the cells were vital and formed distinct columns along the silk fibres. In conclusion, our results show that artificial nerve grafts can be constructed successfully from spider silk, acellularized veins and SC mixed with matrigel.     

Structure,composition and mechanical properties of the silk fibres of the egg case of the Joro spider, <Emphasis Type="Italic">Nephila clavata</Emphasis> (Araneae,Nephilidae)

The silk egg case and orb web of spiders are elaborate structures that are assembled from a number of components. We analysed the structure, the amino acid and fibre compositions, and the tensile properties of the silk fibres of the egg case of Nephila clavata. SEM shows that the outer and inner covers of the egg case consist of thick, medium and thin silk fibres. The silk fibres of the outer cover of the egg case are probably produced by the major and minor ampullate glands. The silk fibres of the inner cover of the egg case from cylindrical glands appears to be distinct from the silk fibres of the major ampullate glands based on their micro-morphology, mole percent amino acid composition and types, and tensile behaviour and properties. Collectively, our investigations show that N. clavata uses silk fibres from relatively few glands in varying combinations to achieve different physical and chemical properties (e.g., color, diameter, morphology and amino acid composition) and functional and mechanical properties in the different layers of the egg case.     

蜘蛛丝的分子结构与力学性能研究

蜘蛛丝尤其是蜘蛛大囊状腺产生的拖丝,具有独特的机械性能,是自然界颇具应用潜力的生物材料。现代分子生物学技术使蜘蛛丝蛋白基因得以克隆,通过高分子物理化学手段方法的利用,有利于揭示蜘蛛丝蛋白质序列、分子结构、以及分子结构和力学性能之间的关系。对不同种类蜘蛛丝蛋白的深入研究,将为基因工程方法人工合成并改造蜘蛛丝成为可能。     

Transglutaminase mediated grafting of silk proteins onto wool fabrics leading to improved physical and mechanical properties

Transglutaminases have the ability to incorporate primary amines and to graft peptides (containing glutamine or lysine residues) into proteins. These properties enable transglutaminases to be used in the grafting of a range of compounds including peptides and/or proteins onto wool fibres, altering their functionality. In this paper we investigated the transglutaminase mediated grafting of silk proteins into wool and its effect on wool properties. A commercial hydrolysed silk preparation was compared with silk sericin. The silk sericin protein was labelled with a fluorescent probe which was used to demonstrate the efficiency of the TGase grafting of such proteins into wool fibres. The TGase mediated grafting of these proteins led to a significant effect on the properties of wool yarn and fabric, resulting in increased bursting strength, as well as reduced levels of felting shrinkage and improved fabric softness. Also observed was an accumulation of deposits on the surface of the treated wool fibres when monitored by SEM and alterations in the thermal behaviour of the modified fibres, in particular for mTGase/sericin treated fibres which, with the confocal studies, corroborate the physical changes observed on the treated wool fabric.     

Bundles of Spider Silk,Braided into Sutures,Resist Basic Cyclic Tests: Potential Use for Flexor Tendon Repair

Repair success for injuries to the flexor tendon in the hand is often limited by the in vivo behaviour of the suture used for repair. Common problems associated with the choice of suture material include increased risk of infection, foreign body reactions, and inappropriate mechanical responses, particularly decreases in mechanical properties over time. Improved suture materials are therefore needed. As high-performance materials with excellent tensile strength, spider silk fibres are an extremely promising candidate for use in surgical sutures. However, the mechanical behaviour of sutures comprised of individual silk fibres braided together has not been thoroughly investigated. In the present study, we characterise the maximum tensile strength, stress, strain, elastic modulus, and fatigue response of silk sutures produced using different braiding methods to investigate the influence of braiding on the tensile properties of the sutures. The mechanical properties of conventional surgical sutures are also characterised to assess whether silk offers any advantages over conventional suture materials. The results demonstrate that braiding single spider silk fibres together produces strong sutures with excellent fatigue behaviour; the braided silk sutures exhibited tensile strengths comparable to those of conventional sutures and no loss of strength over 1000 fatigue cycles. In addition, the braiding technique had a significant influence on the tensile properties of the braided silk sutures. These results suggest that braided spider silk could be suitable for use as sutures in flexor tendon repair, providing similar tensile behaviour and improved fatigue properties compared with conventional suture materials.     

The preparation and applications of functional fibres from crab shell chitin

Novel chitin–silk fibroin fibres and chitin fibres were prepared by an environmental friendly wet-spinning method. Each aqueous solution of sodium chitin (N-acetylchitosan) salt and its blends of silk fibroin in aqueous 14% sodium hydroxide was spun through a viscose-type spinneret into an aqueous 10% sulfuric acid solution saturated with ammonium sulfate (about 43%), and the corresponding white filament was obtained. The tenacity and elongation values of the chitin–silk fibroin filament decreased with an increase of fibroin content up to 33% by weight. A scanning electron microscopy analysis revealed that both the chitin filament and the chitin–silk fibroin (67:33, w/w) filament had vertical strips with faint scale structures on their surfaces. Some applications of these staple fibres were also reported.     

Spider silk fibres in artificial nerve constructs promote peripheral nerve regeneration

Abstract.  Objective : In our study, we describe the use of spider silk fibres as a new material in nerve tissue engineering, in a 20-mm sciatic nerve defect in rats. Materials and methods : We compared isogenic nerve grafts to vein grafts with spider silk fibres, either alone or supplemented with Schwann cells, or Schwann cells and matrigel. Controls, consisting of veins and matrigel, were transplanted. After 6 months, regeneration was evaluated for clinical outcome, as well as for histological and morphometrical performance. Results : Nerve regeneration was achieved with isogenic nerve grafts as well as with all constructs, but not in the control group. Effective regeneration by isogenic nerve grafts and grafts containing spider silk was corroborated by diminished degeneration of the gastrocnemius muscle and by good histological evaluation results. Nerves stained for S-100 and neurofilament indicated existence of Schwann cells and axonal re-growth. Axons were aligned regularly and had a healthy appearance on ultrastructural examination. Interestingly, in contrast to recently published studies, we found that bridging an extensive gap by cell-free constructs based on vein and spider silk was highly effective in nerve regeneration. Conclusion : We conclude that spider silk is a viable guiding material for Schwann cell migration and proliferation as well as for axonal re-growth in a long-distance model for peripheral nerve regeneration.     

Spiders spinning electrically charged nano-fibres

Most spider threads are on the micrometre and sub-micrometre scale. Yet, there are some spiders that spin true nano-scale fibres such as the cribellate orb spider, Uloborus plumipes. Here, we analyse the highly specialized capture silk-spinning system of this spider and compare it with the silk extrusion systems of the more standard spider dragline threads. The cribellar silk extrusion system consists of tiny, morphologically basic glands each terminating through exceptionally long and narrow ducts in uniquely shaped silk outlets. Depending on spider size, hundreds to thousands of these outlet spigots cover the cribellum, a phylogenetically ancient spinning plate. We present details on the unique functional design of the cribellate gland–duct–spigot system and discuss design requirements for its specialist fibrils. The spinning of fibres on the nano-scale seems to have been facilitated by the evolution of a highly specialist way of direct spinning, which differs from the aqua-melt silk extrusion set-up more typical for other spiders.     

Interactions between Spider Silk and Cells – NIH/3T3 Fibroblasts Seeded on Miniature Weaving Frames

Background

Several materials have been used for tissue engineering purposes, since the ideal matrix depends on the desired tissue. Silk biomaterials have come to focus due to their great mechanical properties. As untreated silkworm silk has been found to be quite immunogenic, an alternative could be spider silk. Not only does it own unique mechanical properties, its biocompatibility has been shown already in vivo. In our study, we used native spider dragline silk which is known as the strongest fibre in nature.

Methodology/Principal Findings

Steel frames were originally designed and manufactured and woven with spider silk, harvesting dragline silk directly out of the animal. After sterilization, scaffolds were seeded with fibroblasts to analyse cell proliferation and adhesion. Analysis of cell morphology and actin filament alignment clearly revealed adherence. Proliferation was measured by cell count as well as determination of relative fluorescence each after 1, 2, 3, and 5 days. Cell counts for native spider silk were also compared with those for trypsin-digested spider silk. Spider silk specimens displayed less proliferation than collagen- and fibronectin-coated cover slips, enzymatic treatment reduced adhesion and proliferation rates tendentially though not significantly. Nevertheless, proliferation could be proven with high significance (p<0.01).

Conclusion/Significance

Native spider silk does not require any modification to its application as a biomaterial that can rival any artificial material in terms of cell growth promoting properties. We could show adhesion mechanics on intracellular level. Additionally, proliferation kinetics were higher than in enzymatically digested controls, indicating that spider silk does not require modification. Recent findings concerning reduction of cell proliferation after exposure could not be met. As biotechnological production of the hierarchical composition of native spider silk fibres is still a challenge, our study has a pioneer role in researching cellular mechanics on native spider silk fibres.     

The molecular mobility of water in natural polymers: silk Bombyx mori with a low water content as studied by 1H DQF NMR

The molecular mobility of water in fibres of natural silk (Bombyx mori) was studied by the double-quantum-filtered (DQF) and single-pulse 1H NMR techniques. The results obtained showed a slow motion of water molecules and their strong interaction with silk macromolecules. At different model functions for resonance lineshape in 1H NMR spectra, the influence of signal linewidth on the estimation of relaxation times and cross-relaxation parameters was considered. The observed 1H DQF NMR signal in B. mori silk fibres (BC = 0.065) indicated a local order and anisotropic motion of water molecules, which leads to 1H-1H dipolar interactions in natural silk fibers due to the creation of the second-rank tensors (T(2,+1), T(2,-1)). DQF spectra were the difference of two Lorentzians with different linewidths and were analyzed using the theory of 1H DQF NMR and the data on residual dipolar interactions in systems with the anisotropic mobility of water molecules. The residual dipolar interactions was insignificant and, as the humidity increased (0.18), no DQF-signals and residual dipolar interactions were observed.