鱿鱼皮胶原蛋白医用止血材料的研究

目的 从鱿鱼皮中提取胶原蛋白并研究经化学和物理方法改性的鱿鱼皮胶原海绵的止血效果。方法 从新鲜鱿鱼皮中提取得到酸溶性胶原蛋白 (ASC)和酶溶性胶原蛋白 (PSC),冷冻干燥制备胶原海绵,分别采用EDC交联、干热交联 (DHT)、EDC/DHT结合交联这3种方法对其进行改性处理。采用兔耳创伤止血和肝脏止血试验来评价改性后胶原海绵的止血性能。结果 鱿鱼皮胶原蛋白ASC和PSC为典型的I型胶原蛋白。动物止血实验结果表明ASC-E、PSC-E、ASC-E/D和PSC-E/D等4种改性鱿鱼皮胶原海绵组均能对创面起到一定止血作用,其中尤以PSC-E组的止血效果最好,优于市售明胶海绵。 结论ASC-E、PSC-E、ASC-E/D和PSC-E/D等4种改性鱿鱼皮胶原海绵组均能有效的缩短出血时间,减少出血量,达到快速止血的效果,其中PSC-E止血效果最佳。     

巴沙鱼皮胶原蛋白的提取、组成及变性温度研究

目的 从巴沙鱼皮中提取胶原蛋白并分析其结构类型及氨基酸组成,同时研究物理、化学交联法对其变性温度的影响。 方法 采用酸提法和酶提法提取巴沙鱼皮中的胶原蛋白,并用SDS-PAGE凝胶电泳、FTIR和HPLC分析胶原蛋白的结构类型和氨基酸组成。通过测定粘度值研究物理、化学交联法对其变性温度的影响。 结果 酸提法提取胶原蛋白的提取率为58.2%,酶提法的提取率为22.0%;巴沙鱼皮鱼胶原蛋白为I型胶原蛋白;经120 ℃热交联后,巴沙鱼皮胶原蛋白的变性温度从34.2 ℃下降至28.0 ℃,经四甲基乙二胺(EDC)交联后,其变性温度上升至35.8℃。 结论 巴沙鱼皮富含I型胶原蛋白,酸提法比酶提法提取效率高,采用化学交联法可提高其变性温度,因而巴沙鱼皮可作为胶原蛋白的重要来源,可用于制备多种生物医用材料,具广泛的应用前景。     

3种不同方式提取鳕鱼鱼鳔胶原蛋白与胶原蛋白肽的基本特性研究

目的 研究三种不同提取方式所制备的大西洋鳕鱼（Gadus macrocephalus）鱼鳔胶原蛋白的基本特性。方法 采用低温酸溶提取酸溶性胶原蛋白(acid-soluble collagen, ASC),热水抽提明胶(gelatin, GEL),复合蛋白酶酶解制备鳕鱼鱼鳔肽(swim bladder peptides, SWP)对鳕鱼鱼鳔中胶原蛋白的特性进行研究。对提取的胶原蛋白进行感官评定、SDS-PAGE电泳分析、紫外光谱分析、傅里叶红外光谱分析、氨基酸组成分析与扫描电镜分析。结果 ASC、GEL与SWP三种胶原蛋白的提取率分别为42.15%、72.20%与89.11%,且羟脯氨酸的含量为8.73%、8.97%与7.94%;SDS-PAGE图谱显示ASC至少由两条α链与一条β链组成,GEL中存在少量的α链与β链,SWP种α链与β链被降解。紫外光谱结果显示三种蛋白在230 nm波长处均有最大吸收;扫描电镜结果显示ASC与GEL具有一定的交联结构,SWP中无交联结构存在。结论 三种不同提取方式制备的胶原蛋白具有不同的特性,为鳕鱼鱼鳔胶原蛋白的开发与应用拓宽了领域。     

重组类人胶原蛋白与牛源Ⅰ型胶原蛋白的比较研究

目的比较重组类人胶原蛋白(RHCg)和牛源Ⅰ型胶原蛋白(BTCg)的异同。方法用有限胃蛋白酶水解和盐析沉淀法从牛肌腱中提取纯化BTCg。通过氨基酸分析测定BTCg和RHCg的氨基酸组成;用园二色谱表征BTCg和RHCg在溶液中变性前后的二级结构;用差示扫描量热仪(DSC)、调制差示扫描量热仪(MDSC)以及热重仪(TG)分析BTCg和RHCg的热学性质。结果RHCg和BTCg的氨基酸组成有显著差异。特别是RHCg不含羟脯氨酸;亚氨基酸含量为17.83%(残基数),低于BTCg的22.03%(残基数);赖氨酸含量为0.91%低于BTCg的2.37%;谷氨酸含量为8.13%低于BTCg的12.91%;精氨酸为1.01%低于BTCg的5.33%。在溶液中,温度为20℃时,BTCg和RHCg分子的二级结构均为左旋聚脯氨酸(P-Ⅱ)构型。BTCg和RHCg在140~280℃区间的热学性能显著不同。结论BTCg和RHCg分子的二级结构相似,但氨基酸组成和热学性质存在显著差异。充分认识和理解不同来源胶原蛋白质的特性对于合理设计和使用胶原基生物材料具有重要意义。     

血清酸溶性糖蛋白测定在肾脏疾病中的应用

糖蛋白是一类糖或其衍生物与肽链的氨基酸,以共价健结合的蛋白质,人体血浆蛋白,细胞膜及胶原组织等含有糖蛋白,它在人体内具有广泛的和重要的生理功能。在临床很多疾病的发生过程中,血清酸溶性糖蛋白均有明显改变,本文应用酚试剂显色法测定了正常人血清酸溶性糖蛋白236例,共分两组,即儿童组64人,其中女30人,男34人;儿童组男女无显     

人胎盘来源Ⅳ型胶原蛋白的提取与纯化

目的从人胎盘中分离纯化Ⅳ型胶原蛋白,为制备其抗体提供抗原。方法选择人胎盘为提取原料,用胃蛋白酶消化、氯化钠盐析、凝胶化、超速离心、离子交换层析等方法,提取纯化Ⅳ型胶原蛋白。采用SDS-PAGE对提取的Ⅳ型胶原蛋白进行鉴定。结果胃蛋白酶消化法,使获得胶原蛋白的胶原链部分断裂,凝胶化方法可预先分离出组织中的Ⅰ、Ⅱ、Ⅲ型胶原蛋白,保留了Ⅳ、Ⅴ胶原蛋白,DEAE-Sephrose层析图表明,层析柱吸附的蛋白经洗脱得一主峰,经SDS-PAGE证实提取的Ⅳ型胶原蛋白的分子量80Kd和40Kd两条带。结论从人胎盘中提取的Ⅳ型胶原蛋白纯度高,符合Ⅳ型胶原的特征。提取材料广泛、实验条件简便,结果可靠。     

三重四级杆液质联用测定胶原蛋白海绵中L-羟脯氨酸含量

目的 建立液质联用法测定胶原蛋白海绵中L-羟脯氨酸的方法。方法 采用6 mol·L^-1盐酸-0.5%苯酚酸解胶原蛋白海绵,以乙腈-0.1%甲酸(5∶95)为流动相,质谱测定特征氨基酸L-羟脯氨酸,以茶氨酸为内标定量分析。结果 方法检出限为0.02 ng,线性范围为2.04～30.6μg·m L^-1,方法的重复性RSD为2.4%,准确度为98.3%(RSD=2.3%)。结论 本方法准确可靠,可作为胶原蛋白类产品的含量测定方法。     

鳕鱼皮胶原蛋白的制备及其成分分析

目的 探索如何提高鱼皮胶原蛋白的得率和质量，从而为水产品下脚料的开发利用研究提供参考。方法 采用以酶法为主、热水浸提和酸法浸提为辅的方法提取鳕鱼皮胶原蛋白，对在胃蛋白酶添加量和料液比两参数不同的情况下提取的胶原蛋白得率进行统计，并对所提胶原蛋白进行了成分分析。结果与讨论 胃蛋白酶添加量为1％，料液比为1：10时胶原蛋白得率较高。提取鳕鱼皮胶原蛋白最适宜的胃蛋白酶添加量为1%，料液比为1：10。     

胶原溶液的安全性试验研究

本文对从大鼠尾肌腱用酸溶解法提取的胶原溶液,进行了急性毒性、刺激和过敏等安全性实验.结果表明,胶原溶液是一种无毒、无刺激、无过敏性的安全新型外用药物.     

胶原溶液的安全性试验研究

本文对从大鼠尾肌腱用酸溶解法提取的胶原溶液，进行了急性毒性、刺激和过敏等安全性实验．结果表明，胶原溶液是一种无毒、无刺激、无过敏性的安全新型外用药物。     

Electrophoretic analysis of type I collagen from bovine Achilles tendon: comparison between the extracted raw material and the freeze-dried product

The present study was aimed at verifying if the freeze-drying process has any effect on the polypeptide composition of the collagen extracted from bovine Achilles tendon in an acetic acid gelified form. Data from our laboratories showed that the freeze-drying process renders the collagen gel essentially insoluble; under these conditions the collagen sample can no longer be analyzed by gel electrphoresis. We found that treatment of the sample with pepsin in acid environment, followed by precipitation with ammonium sulphate yields an insoluble fraction that is susceptible of being analyzed by polyacrylamide gel electrophoresis. The The electrophoresis, run under standard conditions, shows that six major subunits, corresponding to alpha, beta and gamma polypeptides, can be revealed in the sample treated in this way. So the freeze-dried collagen exhibits a polypeptidic composition that is basically identical to that shown by the collagen gel, with regard to the fraction precipitated with ammonium sulphate. Otherwise the pattern of the enzymatic hydrolysis was investigated by measuring the hydroxyproline content, and so the collagen content using the 7.46 conversion factor from hydroxyproline to the scleroprotein collagen, in the various steps of the hydrolysis itself: the analytical results showed no differences between the freeze-dried collagen and the gelified form; this confirms that the lyophilization process does not alter the polypeptidic composition of the collagen in any way.     

胶原蛋白凝胶的制备及其中药物体外扩散性考察

目的:提取鱼鳞胶原蛋白,制成凝胶并考察其中药物的体外扩散性。方法:鱼鳞经胃蛋白酶提取、盐析、纯化、冷冻干燥得到胶原蛋白。以水杨酸为模型药物,加入柠檬酸溶液、甘油和乙醇混合液制备胶原蛋白凝胶,并采用琼脂扩散法以扩散系数k为指标评价其体外扩散性,同时与水杨酸水溶性和油脂性软膏进行比较。结果:所得胶原蛋白含量为91.04%;所制水杨酸胶原蛋白凝胶及水溶性和油脂性软膏扩散系数k值分别为68.11、58.04、17.67mm2·h-1。结论:所制凝胶中药物体外扩散速率较快,胶原蛋白可作为载体用于需较快释放药物的外用制剂。     

Demonstration of cellular retinoic acid binding protein (CRABP) in chick embryo tendon cells and effects of retinoids on collagen synthesis in tendon and sterna

The binding of all-trans-retinoic acid (all-trans RA) to specific cytosol proteins and the effects of retinoids on procollagen synthesis were studied in chick-embryo tendon cells. For the receptor assay, tendon cytosols were incubated with [3H]all-trans-RA in the presence or absence of 100-fold excess of nonlabeled all-trans-RA up to 20 hr at +4 degrees and unbound retinoid was removed by charcoal-dextran treatment or by gel filtration chromatography. The results indicated that chick-embryo tendon cells contained cellular retinoic acid binding protein (CRABP). The binding of [3H]all-trans-RA could be displaced by 13-cis-retinoic acid, but not by retinol or etretinate. In contrast no CRABP could be found in cartilage cells isolated from sterna or in whole sterna. The treatment of tendon cytosol with proteases (pronase, trypsin, chymotrypsin) abolished the specific binding of [3H]all-trans-RA. Gel filtration studies on Sephadex G-100 indicated an apparent molecular weight of 14,500-15,000 daltons for the all-trans-retinoic acid binding protein. All-trans-RA markedly decreased procollagen synthesis in isolated chick-embryo tendon cells, the inhibition being concentration dependent; the decrease was about 58% of the control in the presence of 10(-5) M all-trans-RA. Similar decrease was noted with 13-cis-RA and etretinate, while retinol was less effective. In isolated cartilage cells the dose of 10(-5) M of all-trans-retinoic acid decreased drastically total protein and collagen synthesis. The mannosylation of procollagen, the conversion of procollagen to collagen and the size of procollagen chains were not significantly affected. The results of the present study indicate that CRABP is not expressed in sterna of chick-embryos, and in contrast high levels of CRABP could be found in tendons. However, retinoids modulated collagen synthesis in both tissues. Thus it is possible that retinoids can affect the metabolism of different collagen types also in clinical use.     

Characterization of human growth hormone from acromegalic pituitary tumors

Human growth hormone (HGH) was extracted from acromegalic pituitary tumors at pH 10.5 and precipitated with ammonium sulfate at 20–40% saturation. It was purified on a Sephadex G-100 column to yield monomeric HGH. The tumor-HGH was indistinguishable from the authentic one in polyacrylamide gel electrophoresis at pH 8.3 or in the presence of sodium dodecyl sulfate, high-performance liquid chromatography, radioimmunoassay, peptide map, amino acid composition and N-terminal partial amino acid sequence. The tumor-HGH is active in the tibia assay and body weight gain test in hypophysectomized rats with comparable potency to that of the authentic sample.     

Extraction,optimization and characterization of collagen from sole fish skin

In this study, collagen was successfully extracted from marine waste i.e. Sole fish skin, which is available in the coastal area of Mangalore, India. The extraction process was optimized using One Variable at a Time (OVAT) and Response Surface Methodology (RSM) with Box-Behnken Design (BBD) was to achieve maximum yield and the extracted collagen was characterized. The optimal conditions to obtain highest collagen yield was determined to be, an acetic acid concentration of 0.54 M, NaCl concentration of 1.90 M, solvent/solid ratio of 8.97 ml/g and time of 32.32 h. The maximum collagen yield of 19.27 ± 0.05 mg/g of fish skin was achieved under the optimal conditions. The analysis of variance and contour plots exhibited a significant interaction of all the selected variables over collagen extraction process. The SDS-PAGE (Sodium dodecyl sulfate - polyacrylamide gel electrophoresis) analysis suggested that the extracted collagen contained three α-chains i.e. (α1)₂, α2 (M.W. 118, 116 kDa) and one β chain (M.W. 200 kDa) which was similar to commercially available calfskin Type I collagen. FT-IR (Fourier Transform Infrared Spectroscopy) analysis confirmed the existence of helical arrangements of collagen. SEM (Scanning electron microscopy) observation revealed that the extracted collagen was in the form of fibrils with irregular linkages.     

Effect of sodium fluoride on collagen cross-link precursors

Rabbits of similar age and body weight received sodium fluoride (NaF) (50 mg/kg body weight/day) intragastrically for up to 136 days. The acid-soluble collagen of bone, tendon, trachea and skin was extracted and purified. Aldehyde associated with the collagen was determined spectrophotometrically. Bone collagen, which had maximum aldehyde content in normal conditions, showed maximum reduction after sodium fluoride ingestion, as compared to other tissues. The mode of action of fluoride appears variable from tissue to tissue. The collagen fibres produced during fluoride toxicity would be defective due to inadequate cross-links. Thus sodium fluoride interferes with the maturation and normal metabolism of tissue collagen.     

Bioflavonoids effectively inhibit smooth muscle cell-mediated contraction of collagen matrix induced by angiotensin II

Plant-derived bioflavonoids have been recognized to support arterial wall structural integrity and interfere with a variety of proatherosclerotic stimuli. In this study we tested the effects of bioflavonoids on the contractile activity of cultured human aortic smooth muscle cells (SMC) embedded in a 3-dimensional type I collagen matrix. Collagen I solution mixed with human aortic SMC in 24-well plates were allowed to form gels. Tested compounds were added to the wells, and the gels were set afloat by gentle tapping. Digital photographs of the gels were taken after 24 hours of incubation at 37 degrees C. The area of contracted gel was measured and expressed as a percentage of the control gel area from 3 or more replicates. Expression of matrix metalloproteinase (MMP-2) in conditioned media was assessed by gel zymography. Different classes of bioflavanoids showed variable efficiency in inhibiting angiotensin II (ATII)-dependent collagen gel contraction by SMCs. An increase in the number of gallate groups per catechin molecule was associated with increased inhibition of angiotensin II-dependent collagen gel contraction by SMC. Antioxidants (N-acetyl cysteine and ascorbic acid) did not inhibit collagen gel contraction. Bioflavonoid inhibition of collagen gel contraction by SMC correlated with inhibition of matrix metalloproteinase-2 expression. Bioflavonoids participate in the regulation of SMC-mediated contraction and have a strong potential in counteracting pathophysiological effects of ATII. Bioflavonoid activity depends on structural characteristics and can be related to extracellular matrix integrity.     

Review of computer-aided models for predicting collagen stability

Collagen is the most abundant protein in the whole human body and its instability is involved in many important diseases, such as Osteogenesis imperfecta, Ehlers-Danlos syndrome, and collagenopathy. The stability of the collagen triple helix is strictly related to its amino acid sequence, especially the main Gly-X-Y motif. Many groups have used computational methods to investigate collagen's structure and the relationship between its stability and structure. In this study, we initially reviewed the most important computational methods that have been applied in this field. We then assembled data on a large number of collagen-like peptides to build the first Markov chain model for predicting the stability of the collagen at different temperatures, simply by analyzing the amino acid sequence. We used the literature to assemble a set of 102 peptides and their relative melting temperatures were determined experimentally, indicating a great variance with the main motif of the collagen. This dataset was then split in two classes, stable and unstable, according to their melting temperatures and the dataset was then used to build artificial neural network (ANN) models to predict collagen stability. We built models to predict stability at temperatures of 38°C, 35°C, 30°C, and 25°C degrees, and all models had an accuracy between 82% and 92%. Several cross-validation procedures were performed to validate the model. This method facilitates fast and accurate predictions of collagen stability at different temperatures.     

柱前衍生化RP-HPLC测定芜菁子中的12种游离氨基酸

目的采用柱前衍生化PR-HPLC测定芜菁子中的游离氨基酸。探讨芜菁子的药用价值,挖掘新疆的芜菁子资源。方法采用水中超声提取异硫氰酸苯酯柱前衍生化后,用HPLC法测定芜菁子中的氨基酸。结果芜菁子中含有苏氨酸、缬氨酸、脯氨酸、蛋氨酸、甘氨酸、亮氨酸、苯丙氨酸、酪氨酸、丙氨酸、胱氨酸、色氨酸、异亮氨酸等12种游离氨基酸,其中人体必须氨基酸7种,约占总游离氨基酸的65.7%,12种氨基酸在45 min内可很好地分离,平均回收率为92.7%~98.2%。结论所建方法不需专门的氨基酸分析仪,操作简便、灵敏度高、准确可靠。芜菁子中氨基酸的种类齐全,含量丰富,具开发价值。