鱼鳞中胶原蛋白的提取纯化及应用

总结了鱼鳞中胶原蛋白的提取、分离和纯化方法及其结构特点、生物学功能,并探讨了其在食品、生物、医学、造纸、饲料等方面的应用及发展前景。     

胶原蛋白在组织工程及临床中的应用

1.前言 胶原蛋白(以下简称为胶原)呈纤维状,是哺乳动物中含量最为丰富的动物蛋白。胶原能与各种细胞结合以构成性能各异的组织,如:软骨、皮肤、骨骼和肌腱。由于胶原是构成哺乳动物和人类众多组织的成份之一,所以近年来成为一种在组织工程上应用十分广泛的生物材料。尽管其研究的面涉及范围较广,但其主要有三个方面:①分子结     

天然降解性生物材料在整形外科中的应用

整形外科与生物医用材料有着密切的关系。互为依存且互促发展,本文将近年来新型的生物医用材料结合本研究所多年来的科研。重点阐述胶原蛋白,透明质酸及医用几丁糖的基本情况,并努力探索他们在整形外科中可能的应用。     

重组人源性胶原蛋白的制备及表征

为了获得可实现工业化生产的重组人源性胶原蛋白,根据人I型胶原蛋白Gly-X-Y序列,优选亲水性的Gly-X-Y胶原肽段设计人源性胶原蛋白氨基酸序列及对应的核苷酸序列,利用酶切技术构建pPIC9K-COL表达载体,电转化毕赤酵母获得人源性胶原蛋白毕赤酵母工程菌,并对其进行发酵罐发酵、纯化及鉴定。结果显示,获得表达量达4.5 g/L,纯度大于95%的人源性胶原蛋白,经氨基酸N端测序、分子量测定、氨基酸分析及胶原酶降解试验,确定获得的蛋白与理论的人源性胶原蛋白一级结构一致;同时胶原经冷冻干燥后进行扫描电镜分析及细胞毒性试验,确定人源性胶原蛋白冻干品具有多孔纤维网状结构及优良的细胞相容性,预示其具备作为生物医学材料的潜质。     

海蜇胶原蛋白的提取及纯化(英文)

通过胃蛋白酶的限制性酶切、选择性盐析及阳离子交换柱的层析作用,低温从海蜇中胶层提取可得到胃蛋白酶促溶且去除端肽(telopeptide)的胶原蛋白(PSC)。紫外-可见扫描测得PSC的最大吸收峰位于235nm。凝胶电泳及氨基酸分析显示这些都符合Ⅰ型胶原的特征,同水母S.nomurai和Rhopilema asamushi来源的胶原蛋白基本一致,作为一个低等动物,其氨基酸分析显示了脊椎动物和非脊椎动物的微小差异。     

中华鳖多糖制备纯化及一般理化性质研究

利用双酶酶解工艺制备了中华鳖体内的多糖物质,以乙醇沉淀纯化了鳖多糖。鳖多糖为灰白色粉未状,易溶于水,不溶于丙酮等有机试剂,其中多糖质量分数为82.12%,它的蛋白、硫酸根及已糖醛酸质量分数分别是3.20%、2.16%、10.36%。     

乌贼皮胶原蛋白的提取及结构表征

为了充分利用乌贼加工废弃物,分析了乌贼皮的基本组成成分,优化了从乌贼皮中提取胶原蛋白的工艺条件,并利用SDS-PAGE垂直电泳、紫外扫描和傅里叶变换红外光谱对所提取的胶原蛋白进行了结构表征.结果表明,乌贼皮中含有大量胶原蛋白,可作为胶原蛋白来源的补充.采用酸酶复合提取胶原蛋白的最佳条件为:酒石酸浓度为0.1mol/L,胃蛋白酶添加量为1400U/g,料液比为1:20(m:V,原料),4℃提取18h,提取率为12.08％.SDS-PAGE垂直电泳、紫外扫描和傅里叶变换红外光谱的结果表明,采用酸酶复合法从乌贼皮中提取的胶原蛋白为I型胶原蛋白,保持了完整的三螺旋结构.     

鼠尾胶原蛋白提取、分离、纯化方法的建立及鉴定

目的建立一种高效提取、分离、纯化鼠尾胶原蛋白的方法。方法通过对鼠尾进行剥离获得鼠尾腱,用Tris-HCl缓冲液、胃蛋白酶处理获得鼠尾胶原蛋白原液、反复使用氯化钠溶液进行分级盐析、醋酸溶液复溶进行鼠尾胶原蛋白的纯化。超纯水透析除去无机盐类获得纯化的鼠尾胶原蛋白。通过SDS-PAGE蛋白质电泳、氨基酸含量分析等技术手段鉴定。结果本研究建立的方法可以获得高纯度的鼠尾胶原蛋白,纯度达到电泳纯。与国外进口的商业化鼠尾胶原蛋白产品相比无差异。研究了提取、分离、纯化参数对得率、纯度的影响,建立了最优的鼠尾胶原蛋白提取条件,胃蛋白酶用量：1∶500,酶解时间：72 h,盐析浓度：2 mol/L,提取所用酸溶液：0.05mol/L醋酸溶液。结论为鼠尾胶原蛋白的扩大化生产提供了合适的工艺参数,为大量获得鼠尾胶原蛋白并进行更深层次的功效方面研究提供了理论支持和实践基础。     

葛根素的提取及纯化技术

葛根素是葛根的主要活性成分 ,因其对许多疾病有良好的治疗作用而日益受到广泛的关注。本文主要论述了从葛根中提取纯化葛根素的工艺方法     

Morphologic analysis of atretic follicles in the Chinese soft-shelled turtle,Pelodiscus sinensis

The Chinese soft-shelled turtle (Pelodiscus sinensis) and its eggs have been utilized as both a source of nutrition and medicine for thousands of years. In the present study, follicular atresia was investigated by morphological analysis as an important factor affecting clutch size. Results showed that follicular atresia can be divided into four stages: (1) development of follicular cell disorder; (2) massive cellular proliferation in the ooplasm with elimination of yolk granules; (3) large amounts of erythrocytes appearing in the ooplasm; (4) gradual degeneration of atretic follicles. For the first time, we have shown follicular atresia to be a dynamic process in P. sinensis and found that yolk granules are eliminated by both liquefaction as well as non-liquefaction processes. The present study may provide detailed morphologic analysis for further research into egg quality and clutch size in P. sinensis.     

Aphanomyces infection in juvenile soft-shelled turtle,Pelodiscus sinensis,imported from Singapore

Aphanomyces sp. was isolated from the carapaces of two juvenile soft-shelled turtles with fungal infections imported from Singapore. Their sizes were 2.9–3.5 cm in carapace length. Lesions with integumental necrosis and ulceration looked like white cotton. The fungus exhibited slow growth, hyphae were 7.5–15 μm in diam, coarse, and abundantly branched. Zoosporangia observed in the isolate were complex, its entire thallus being converted into zoosporangial units, with short or long lateral evacuation tubes, and isodiametric, 100–500 μm in length. Clusters of zoospores were also produced at the terminals of hyphae. The production of the primary zoospores was achlyoid. The primary encysted zoospores were spherical, 10–15 μm in diam. No sexual stages were observed on a hemp seed incubated in sterile tap water. The optimal temperature for the fungus was 30° C.     

中华鳖精子头部的形成

应用透射电镜技术详细研究了中华鳖精子头部形成过程的超微结构变化。结果显示,中华鳖精子头部的形成过程可分成5个连续时期：第1期前顶体泡形成并移向细胞核一侧,同侧核膜凹陷成浅窝。前顶体泡底部中央出现小的顶体颗粒,纤维物质层位于核前端与前顶体泡底壁之间,其核膜一侧的中央形成更小的顶体下颗粒,将与核内小管的形成有关。细胞核开始端移和变形。第Ⅱ期核浅窝逐渐外推,前顶体泡变成扁囊状覆盖于隆突的核顶端,顶体颗粒弥散成中等电子致密物分布于顶体帽中,纤维物质层发育为顶体下锥。环形核套微管在顶体后端的核周围逐渐形成,核内染色质开始浓缩成圆形颗粒,核膜下出现明显问隙,细胞核体积变小。顶体下颗粒消失,但其下端的核质中可见2-4条核内小管开始发生。第Ⅲ期拉长的细胞核前端突出于精子细胞外,表面有顶体复合体覆盖,核后端最宽并出现植入窝。染色质进一步浓缩,颗粒间隙变小,细胞核更细长。第Ⅳ期染色质浓缩成致密均质物,核肩之前的细胞核变细,成为核前突。环形核套微管先后改建为斜行和纵行核套微管,支持细胞突起形成“袖领”包绕顶体。第Ⅴ期核套微管解聚而消失,顶体周围的“袖领”也消失,顶体下间隙出现。结果显示中华鳖精子头部的形成过程,即核质浓缩的形态变化过程、顶体的形成和核内小管的发育与变化方面,存在许多与其他爬行动物不同之处。     

中华鳖精子的超微结构

爬行纲在动物进化中占有重要地位,而且爬行纲动物种类繁多,生态环境和生活习性差异颇为明显。关于爬行动物生殖特性的资料很有限,而且由于动物种类的复杂性,不同动物的研究结果差异较大(Gustavo et al.,2004),缺乏统一的结构模式。精子的形态结构与动物的生殖特性直接相关,在一     

慢性氨暴露对中华鳖幼鳖血浆总氨氮、皮质酮浓度及组织氨代谢酶活性的影响

本文观测了慢性氨暴露对中华鳖(Pelodiscussinensis)幼鳖血浆总氨氮、皮质酮浓度及与氨代谢有关的酶活性的影响。将中华鳖暴露在总氨氮(TAN)浓度为32.4、57.6、83.5mg/L(分别记为1、2、3组)的水环境中饲养42d,以自然晾晒脱氯自来水饲养组为对照(记为0组),pH值控制在7.80-7.85。检测氨暴露2、4、8、24、48h、42d后血浆TAN与8、24、48h、42d后皮质酮浓度,42d后肝、肌肉与脑中谷氨酰胺合成酶(Glutaminesynthetase,GS)、谷氨酸脱氢酶(Glutamatedehydrogenase,GDH)的活性以及特定生长率(Specificgrowthrate,SGR)的变化。结果表明:0、1组血浆TAN随时间没有明显变化,2、3组血浆TAN随时间呈现先增加后降低的趋势,分别在8h和48h达到峰值,42d时各组间血浆TAN没有显著差异。氨暴露显著影响暴露初期血浆皮质酮水平,暴露后24h,1、3组血浆皮质酮水平显著高于对照组;42d后除2组外其他组间无显著差异。4组之间肝、肌肉和脑中GS活性均没有显著差异,肝和肌肉GDH(氨化和去氨化方向)活性也没有显著差异。各暴露组脑GDH活性和对照组相比差异不明显,但各处理组间氨化和去氨化方向脑GDH酶活差异显著。42d饲养期间各处理组SGR没有显著差异。     

Seasonal effects on apoptosis and proliferation of germ cells in the testes of the Chinese soft-shelled turtle, Pelodiscus sinensis

To elucidate the processes involved in the spatial and temporal maturation of spermatogenic cells in the testes of the soft-shelled turtle, Pelodiscus sinensis, we used a histological morphology method, TdT-mediated dUTP nick end-labeling (TUNEL) assay, the proliferating-cell nuclear antigen (PCNA), and electron microscopy. Seminiferous tubules from 100 turtles, normal for size of testes and semen quality, were collected during 10 months of a complete annual cycle (10 turtles/month). The seminiferous epithelium was spermatogenically active through the summer and fall, but quiescent throughout the rest of the year; germ cells progressed through spermatogenesis in a temporal rather than a spatial pattern, resulting in a single spermatogenic event that climaxed with one massive sperm release in November. The TUNEL method detected few apoptotic cells in spermatogenic testis, with much larger numbers during the spermatogenically quiescent phase. Spermatocytes were the most common germ cell types labeled by the TUNEL assay (a few spermatogonia were also labeled). Apoptotic spermatocytes had membrane blebbing and chromatin condensation during the resting phase, but not during active spermatogenesis. We inferred that accelerated apoptosis of spermatogonia and spermatocytes partly accounted for germ cell loss during the nonspermatogenic phase. The PCNA was expressed in nuclei of spermatogonia and primary spermatocytes during the spermatogenically active phase. During the regressive phase, PCNA-positive cells also included spermatogonia and spermatocytes, but the number of positive spermatocytes was less than that during the spermatogenically active phase. We concluded that seasonal variations in spermatogenesis in the soft-shelled turtle were both stage- and process-specific.     

一株枯草芽孢杆菌的分离鉴定、生物学特性及其对水质净化的作用

[背景]随着水产养殖业的发展和养殖集约化程度的提高,养殖水环境日趋恶化,养殖动物病害频发,而水产益生菌因其环境友好、安全而被广泛应用于水产养殖中.[目的]从南美白对虾养殖池底泥分离枯草芽孢杆菌,探究其体外生物学特性及对水质的净化作用,以期扩充微生态制剂的种质资源.[方法]采用稀释涂布平板法分离菌株,通过形态学观察、生理...     

Isolation, purification, and properties of boar sperm cytochrome oxidase

Cytochrome oxidase was isolated from the midpiece of boar sperm by extraction and fractionation with ammonium sulfate in the presence of cholate. The enzyme was further purified to apparent homogeneity by DEAE-cellulose column chromatography in the presence of minimal amounts of Triton X-100. The purified enzyme exhibited similar oxidized and reduced optical spectra to those of the bovine heart and rat liver cytochrome oxidases. However, the sperm oxidase was found to contain stoichiometrically more subunits I, II, and III than the other, smaller subunits. The sperm oxidase also required phospholipids for its activity, but it was less sensitive to inhibition by KCN. Interestingly, the sperm oxidase was much more acid-stable than the bovine heart and rat liver counterparts. The optimumpH for the sperm oxidase catalyzing the electron transfer between ferrocytochromec and cytochromea was aroundpH 4.8, and those for the bovine heart and rat liver were 6.2 and 6.8, respectively. AtpH 4.5, the sperm oxidase still maintained about 70% enzyme activity, whereas less than 20% activity remained in the heart and liver oxidases. The peculiar properties of sperm cytochrome oxidase may be due to the fact that the well-packed chromosomes in the sperm headpiece do not function, such that the nuclear gene-coded subunits are deficient in the sperm cytochrome oxidase. The finding that the sperm oxidase was more acid-stable is an example of structure-function coordination, and is discussed from the viewpoint of chemiosmotic theory and the unique structure and functions of the sperm mitochondria.