Silkworm silk biomaterials for spinal cord repair: promise for combinatorial therapies

Background：Traumatic injury to the adult mammalian spinal cord results in minimal axonal regrowth,cystic cavity formation at the injury site,poor functional recovery and there is no cure available.Due to the complex nature of spinal cord injury（SCI）,a combination of therapeutic strategies may offer the most promise for successful regeneration（Ahuja et al.,2017）.

Super-strong and Intrinsically Fluorescent Silkworm Silk from Carbon Nanodots Feeding

Fluorescent silk is fundamentally important for the development of future tissue engineering scaffolds.Despite great progress in the preparation of a variety of colored silks,fluorescent silk with enhanced mechanical properties has yet to be explored.In this study,we report on the fabrication of intrinsically super-strong fluorescent silk by feeding Bombyx mori silkworm carbon nanodots(CNDs).The CNDs were incorporated into silk fibroin,hindering the conformation transformation,confining crystallization,and inducing orientation of mesophase.The resultant silk exhibited super-strong mechanical properties with breaking strength of 521.9±82.7 MPa and breaking elongation of 19.2±4.3%,improvements of 55.1%and 53.6%,respectively,in comparison with regular silk.The CNDs-reinforced silk displayed intrinsic blue fluorescence when exposed to 405 nm laser and exhibited no cytotoxic effect on cells,suggesting that multi-functional silks would be potentially useful in bioimaging and other applications.

Silkworm Silk Fibers with Multiple Reinforced Properties Obtained through Feeding Ag Nanowires

Silkworm silk fbers have been woven into textiles for thousands of years,because of their attractive luster,good mechanical properties,excellent biocompatibility,and large-scale production.With the development of human society,preparation of silk fbers with modifed or enhanced properties are highly desirable for potential applications in structural materials and smart textiles.Herein,we realized the reinforcement of multiple properties of silk fbers by feeding silkworms with Ag nanowire(Ag NW)modifed diets.The obtained silk fbers show obviously enhanced comprehensive mechanical properties,including improved tensile strength,elongation at break,tensile modulus,and toughness,which are increased by 37.2%,37.6%,68.3%,and 69.8%,respectively.Furthermore,compared with unmodifed silk,the electrical conductivity and thermal conductivity of modifed silk fbers are improved by 246.4%and 32.1%,respectively.The analysis on the components and structure shows that the incorporated Ag NWs lead to increased content of random coil/α-helix,improved orientation of crystallites,and increased content of Ag compared to pristine silk fbers,which may contribute to the enhanced mechanical,electrical,and thermal properties.

Mechanical properties of cocoons constructed consecutively by a single silkworm caterpillar,Bombyx mori

Most animals have the ability to adapt, to some extends and in different ways, the variation or disturbance of environment. In our experiments, we forced a silkworm caterpillar to spin two, three or four thin cocoons by taking it out from the cocoon being constructed. The mechanical properties of these cocoons were studied by static tensile tests and dynamic mechanical thermal analysis. Though external disturbances may cause the decrease in the total weight of silk spun by the silkworm, a gradual enhancement was interestingly found in the mechanical properties of these thin cocoons. Scanning electron microscopy observations of the fractured specimens of the cocoons showed that there exist several different energy dissipation mechanisms occurred simultaneously at macro-, meso-, and micro-scales, yielding a superior capacity of cocoons to adsorb the energy of possible attacks from the outside and to protect efficiently its pupa against damage. Through evolution of millions of years, therefore, the silkworm Bombyx mori seems to have gained the ability to adapt external disturbances and to redesign a new cocoon with optimized protective function when its first cocoon has been damaged for some reasons.

Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bombyx mori) on Day 1 and Day 4 in the 5th Instar Stage

The posterior silk gland （PSG） of silkworm is an important organ where fibroin is synthesized and secreted exclusively. Because fibroin constitutes 75-80% of the silk filament, the mechanism governing fibroin secretion, quality and yield of cocoon can be elucidated by the study on the PSG. Using two-dimensional gel electrophoresis （2-DE） and image analysis system, the changes in the protein composition in the PSG cell were investigated on the day 1 （D1） and day 4 （D4） in the 5th instar stage from five different strains of silkworm （Bombyx mori）. While differences at protein level between days and strains were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse among the different strains. The results suggest that the secretion of fibroin is regulated by multiple proteins. The site of regulation and the proteins responsible for the regulation vary with the strain, which leads to differences between strains in the capacity of fibroin secretion in the PSG cell.

Analysis of Protein Expression Patterns of Silkworm Jinqiu and Its Cross Parents

The differences of protein expression between the improved cross breeding race Jinqiu and its parents were analyzed to discuss the gene construction, and to form a base for illuminating the molecular mechanisms of successful cross breeding in silkworm. Protein samples from silk gland, hemolymph, and midgut were separated by 2-dimensional gel electrophoresis （2-DE）. In the three tissues the matched protein spots between Jinqiu and its cross parents were approximately 70% with approximately 30% specific protein spots. In the matched protein spots, 9-24% was differentially expressed representing up- and down-regulated expression. These specific protein spots might be either the newly appeared, which were produced from the genic interaction of cross parents＇ genes in cross breeding, or posttranscriptionally modified, which were produced from the different modifications on the same original proteins. These results indicate that it is important for a new successful breed, by cross breeding, relying on the actions of some newly produced functional proteins from genic interaction, in addition to marshaling excellent genes of cross parents.

Highly efficient expression of human extracellular superoxide dismutase(rhEcSOD)with ultraviolet-B-induced damage-resistance activity in transgenic silkworm cocoons

Extracellular superoxide dismutase(EcSOD)protects tissues from oxidative stress,and thus is considered as a therapeutic agent for many diseases such as atherosclerosis,hypertension,and cancer.However,cost-effective production of bioactive recombinant human EcSOD(rhEcSOD)remains a challenge.Herein,we developed an efficient strategy for producing active rhEcSOD by transgenic silkworms.rhEcSOD was successfully synthesized as homodimers and homotetramers in the middle silk gland and spun into the cocoons with a concentration of 9.48±0.21 mg/g.Purification of rhEcSOD from the cocoons could be conveniently achieved with a purity of 99.50%and a yield of 3.5±0.5 mg/g.Additionally,N-glycosylation at the only site of N89 in rhEcSOD with 10 types were identified.The purified rhEcSOD gained the potent enzymatic activity of 4162±293 U/mg after Cu/Zn ions incorporation.More importantly,rhEcSOD was capable of penetrating and accumulating in the nuclei of cells to maintain cell morphology and attenuate ultraviolet B-induced cell apoptosis by eliminating reactive oxygen species and inhibiting the C-Jun N-terminal kinase signaling pathway.These results demonstrated that the transgenic silkworm could successfully produce rhEcSOD with enzymatic and biological activities for biomedical applications.

桑蚕的强制牵伸抽丝及其纤维性能

为提升蚕丝的力学性能,采用一种圆锥斜面牵伸装置对五龄桑蚕进行强制牵伸抽丝以制备高强牵伸丝,研究不同牵伸速度对桑蚕牵伸丝形貌、力学性能的影响,并将牵伸丝与蚕丝进行对比与分析。结果表明:由强制抽丝获得的牵伸丝表面光滑平坦,丝胶均匀附着;随着牵伸速度的增加,牵伸丝截面逐渐向弓形转变;牵伸丝的综合力学性能显著优于蚕丝,且其断裂强度、弹性模量、断裂比功与断裂伸长率随着牵伸速度的增加均有不同程度的增加;其中,以接近桑蚕自然吐丝速度(1 cm/s)牵伸制备的牵伸丝的断裂强度与蚕丝相当,约为3.00 cN/dtex,但其弹性模量(93.10 cN/dtex)远高于蚕丝(76.90 cN/dtex);当牵伸速度增加至4 cm/s时,可获得力学性能最佳的牵伸丝,其断裂伸长率为22.49%,断裂强度为3.39 cN/dtex,弹性模量为95.76 cN/dtex,断裂比功为0.62 cN/dtex,相比于蚕丝的平均值分别提高了7%、13.76%、24.53%、31.91%。

Preparation and Characterization of PEGDE Crosslinked Silk Fibroin Film

To obtain water-insoluble silk fibroin （SF） materials, polyethylene glycol diglycidyl ether （PEG-DE） was selected as a crosslinking agent to prepare SF films （blends）. The reaction conditions were optimized for the crosslinking of the SF molecules. The hot water stability of the blends was measured using BCA protein assay and gravimetric analysis. The molecular conformation and crystalline structure of the blends were analyzed by FTIR and XRD, respectively. When the mass ratio of SF：PEG-DE was 1.0：0.8, the hot water loss rate of the SF blends was minimized. PEG-DE could induce SF molecules to form fl-sheets during the gel reaction process, resulting in improved crystallinity and hot water dissolved resistance of the blend films. In order to demonstrate the eytotoxicity of the chemical reagents used to crosslink SF, L929 cells were seeded on the blend film （SF：PEG-DE = 1：1） and cultured for 3 days. Cells of L929 readily adhered and spread in the fusiform on the blend film resulting in high cell viability. The extracted liquid from the SF porous film did not inhibit cell proliferation, as estimated by the MTT assay.

柞蚕新品种墨玉及其杂交种辽蚕988的缫丝性能测定

通过对柞蚕新品种墨玉及其杂交种辽蚕988的缫丝性能测定,明确了新品种的缫丝性能。柞蚕新品种墨玉雌茧的茧丝长1533.6 m、丝重1.03 g、纤维公量1.36 g,分别比对照种三里丝高19.9%、25.61%和20.35%,与对照种抗大的8个缫丝性状相当或略优于对照种;雄茧的茧丝长1520.1 m、丝重0.95 g、纤维公量1.26 g,分别比对照种三里丝高20.62%、26.67%和26%,与对照种抗大的缫丝性能相当。杂交种辽蚕988除在茧丝纤度与茧丝量方面略低于墨玉×9906外,其余缫丝性状与对照种抗大×9906及杂交组合墨玉×9906之间均无显著性差异。新品种墨玉及其杂交种辽蚕988的鲜茧出丝率均超过7%,达到了缫丝性能评价的优级标准。

丝绸字义文化的研究

丝绸,除了产业生产和经济活动以外,还蕴涵着文化韵味。本文列举了丝绸的字义解释,人文典故、织物术语、成语谜语和文学诗词等丝、绸相关文化知识,期望通过丝绸字义的探讨,增进蚕丝绸文化研究内涵与空间,作为蚕丝文化的微补。

角蛋白替代大豆蛋白养蚕对蚕丝结构与性能的影响

角蛋白主要存在于动物的毛发中,经过处理被广泛用作动物蛋白饲料。为探究角蛋白替代大豆蛋白养蚕对蚕丝结构与性能的影响,文中以基础家蚕人工饲料养蚕为对照,分别用酶解角蛋白替代1.0%、2.5%、5.0%、7.5%、10.0%大豆蛋白饲养5龄大蚕。结果表明:随着角蛋白替代比例增加,熟蚕体重、全茧量和茧层量逐渐减少,但替代组茧层率平均提高了8.97个百分点。与对照组蚕丝相比,替代组蚕丝的基本结构并未发生改变,但α螺旋/无规卷曲和相对结晶度有所增加,蚕丝力学性能也有所提升。2.5%替代组蚕丝断裂强度和断裂伸长率比对照组分别提高了8.15%和4.49个百分点,且替代比例≤5.0%不会影响蚕丝纤度。该研究可为角蛋白用作家蚕人工饲料原料提供一定的参考。

不同品种桑蚕牵伸丝的结构与性能

利用自主研制的圆锥斜面强制牵伸抽丝装置对不同品种五龄桑蚕进行强制抽丝,通过拉伸测试、傅里叶红外光谱及X射线衍射对不同品种牵伸丝与相应原茧丝的结构与性能进行表征与分析。结果表明:两种牵伸丝的力学性能均优于其对应茧丝,黄色雄蚕牵伸丝的力学性能更佳,其初始模量、断裂强度、断裂比功与伸长率的平均值分别为117.35、4.55、0.75 cN dtex与26.54%。牵伸作用会导致丝纤维晶粒尺寸减小、结晶度提高,从而赋予丝纤维更为优异的力学性能。

纳米颗粒添食育蚕改性蚕丝纤维的染色性能

为了探究纳米颗粒添食育蚕改性蚕丝纤维的染色性能,选用2%质量分数的纳米二氧化钛和纳米氧化铜添喂家蚕,将蚕茧脱胶后进行染色并测定上染率、色深值等指标,同时探究了酸性橙Ⅱ对蚕丝纤维的染色动力学和热力学机制。结果表明:纳米颗粒添食能改善蚕丝纤维的染色性能;酸性染料和活性染料对改性蚕丝纤维的上染率和表观色深(K S值)均高于空白组,洗涤20次后K S值仍高于空白组1.5和2.0。染色动力学和热力学分析表明酸性橙Ⅱ对各组蚕丝纤维上染过程均符合准二级吸附动力学方程和朗格缪尔(Langmuir)吸附,且属于放热过程,较低温度下更适合染色;60℃下纳米TiO 2和纳米CuO改性蚕丝纤维的实际吸附平衡饱和量分别比空白组高了0.14 g kg和1.04 g kg,计算得到染料吸附的最大值分别比空白组高了5.31 g kg和14.58 g kg,显示了改性蚕丝纤维更优的染色性能。

黄帝“淳化”“虫蛾”成就与嫘祖“先蚕”“蚕神”名号

《史记》卷一《五帝本纪》所记载黄帝“淳化”“虫蛾”成就,显示了早期文明进程中丝绸生产的意义。而“嫘祖为黄帝正妃”,“嫘”的文字学分析,所谓“细丝曰糸”反映了蚕桑业的发明。黄帝事迹,其中有“嫘祖”的表现。《礼记》关于“先蚕”纪念的礼俗,体现当时对这种产业开创功绩的尊重与崇敬。现在看来,黄帝“淳化”“虫蛾”成就作为帝业基础与文明标志,有嫘祖之功的因素。后世“先蚕”“蚕神”名号,亦体现了黄帝、嫘祖在丝绸史上的重要地位。

金纳米三角片对家蚕生长状况和蚕丝性质的影响

本研究通过足部注射的方式将金纳米三角片导入5龄家蚕体内,研究了金纳米三角片对家蚕生长状况以及蚕丝性质的影响。结果表明,金纳米三角片的摄入大幅度降低了家蚕的成活率。总体来看,家蚕平均体质量、体长较对照组有所降低,但外貌无显著变化。X射线衍射(XRD)和红外光谱(FTIR)结果表明,与对照组相比,摄入金纳米三角片的试验组蚕丝的晶体或二级结构并未遭到破坏。该研究可以为金纳米三角片的生物安全性评估提供了一定参考。

天然黄色家蚕丝纤维与其他蚕丝纤维的形态结构比较

为了进一步促进彩色蚕丝的开发和应用,研究了通过杂交育种获得的天然黄色家蚕茧丝的外观形态和截面结构特征,并与普通白色家蚕丝、柞蚕丝和来自柬埔寨的多化性黄茧家蚕丝进行了比较研究。结果显示:天然黄色家蚕丝纤维的横截面形态主要呈三角形,与普通白色家蚕丝、多化性黄茧家蚕丝纤维的横截面形态相似,而柞蚕丝的横截面形态呈长椭圆形。天然黄色家蚕丝的颜色鲜艳,光泽感优于普通白色家蚕丝和柞蚕丝。离子刻蚀显示,天然黄色家蚕丝纤维内部的原纤表面组织结构比普通白色家蚕丝紧密。

转基因家蚕生产含蜘蛛丝蛋白的新型复合茧丝纤维

为了人工大量生产蜘蛛丝蛋白,蜘蛛牵引丝基因(MaSp1)被用piggyBac转基因载体导入家蚕染色体中。茧丝蛋白分析显示转基因家蚕可以生产含有蜘蛛牵引丝蛋白的复合茧丝新纤维。与天然茧丝相比,转基因家蚕茧丝强度增加5.3%,而断裂伸长几乎没有变化。研究表明利用转基因家蚕大量生产各种新型复合蛋白质纤维材料具有潜在的应用前景。

转蜘蛛拖牵丝蛋白基因家蚕蚕丝氨基酸组成及其机械性能

将以绿色荧光蛋白基因为报告基因、含有人工合成的1.6 kb的蜘蛛拖牵丝蛋白基因及转座子pig-gyBac的转基因载体成功导入减秋无滞育家蚕受精卵,得到转蜘蛛拖牵丝蛋白基因家蚕及绿色荧光茧。对转基因家蚕与对照家蚕丝素蛋白进行了氨基酸组成分析,结果表明转基因蚕茧丝素蛋白甘氨酸和丙氨酸的百分含量分别增加了1.65%和1.80%(平均值);对其生丝的机械性能进行了测试研究,结果表明转基因蚕茧生丝的伸长率降低,断裂强度和初始模量增加,且差异均显著,与理论预期结果吻合。结果表明转基因家蚕蚕丝的机械性能一定程度上得到了提高。

微波辅助酸水解法制备丝素肽的研究

[目的]建立并优化快捷、高效制备丝素肽的微波辅助酸水解工艺。[方法]以家蚕废丝为原料,比较在碱液脱胶、磷酸酸解、石灰水沉淀除盐、活性炭脱色的工艺流程中酸水解的优化方案,并探讨亚硫酸和磷酸混合酸解对后继脱色工段的影响。[结果]微波辅助磷酸水解丝素蛋白最佳工艺条件为:磷酸浓度85%,温度70℃,功率150W,酸料比4∶1,水解90min,产品回收率达到69.0%,比磷酸水浴水解方案高约15%,且节省了4～5h的水解时间;在微波辅助水解条件下,亚硫酸和磷酸混合酸解比单独用磷酸酸解所得水解液颜色浅,色值可降低65.46%。[结论]对于丝肽的酸水解法制备,微波辅助能大大降低能耗,节省成本;混合酸酸水解法能降低酸解液色值。