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。     

鼻窦炎行鼻内镜外科手术后不同鼻腔填塞物应用效果分析

目的：探讨鼻窦炎行鼻内镜外科手术后不同鼻腔填塞物应用效果。方法选取2012年～2013年行鼻内镜手术的鼻窦炎患者80例为研究对象,在术后分别应用了凡士林纱条（A组）、PVF医用海绵（B组）、凡士林纱条和胶原蛋白海绵（C组）、PVF医用海绵和消炎抗菌可溶止血纱布（D组）进行术后鼻腔填塞,4组病例各为20例,对比分析4组患者术后疼痛情况与止血情况。结果相对于A组,B组、C组以及D组疼痛率较低,且出血率也较低,差异具有统计学意义（P＜0.05）;B、C、D3组组间比较,差异无统计学意义（P＞0.05）。结论临床中应结合病患自身的实际情况,灵活应用PVF医用海绵、凡士林加用胶原带白海绵或者PVF医用海绵和消炎抗菌可溶止血纱布等,提高病患舒适度,提高止血效果。     

可吸收胶原蛋白止血海绵在腔镜甲状腺手术中的止血疗效

目的总结可吸收胶原蛋白止血海绵在腔镜甲状腺手术中止血疗效的临床经验。方法 2011年2月~2013年2月,对128例接受腔镜甲状腺手术的患者,随机分成观察组和对照组,观察组于术后用可吸收胶原蛋白止血海绵,对照组于术后不用任何止血材料,分别于术后12、24、48h观察甲状腺残腔引流管的引流量。结果观察组术后12、24、48h的引流量均少于对照组(P0.05)。结论可吸收胶原蛋白止血海绵在腔镜甲状腺手术中具有安全、有效的止血效果。     

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。     

鱿鱼皮胶原蛋白水解肽抗氧化活性研究

目的探讨秘鲁鱿鱼(Dosidicus eschrichitiiSteenstrup)皮胶原蛋白多肽组分对自由基的清除作用以及水解产物的体内抗氧化作用,为鱿鱼加工废弃物的高值化利用探索一条新途径。方法采用化学发光法研究胶原蛋白活性多肽体外对超氧阴离子自由基(O2.-)和羟自由基(.OH)的清除作用,并筛选出体外清除自由基活性最好的组分;灌胃于D-半乳糖致衰老的模型小鼠,测定小鼠血液及皮肤中的超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活力和丙二醛(MDA)及羟脯氨酸(Hyp)含量。结果与结论鱿鱼皮胶原蛋白多肽分子量小于2000D组分对O2.-和.OH具有较好的清除效果,该活性多肽组分可以提高小鼠血液及皮肤中SOD和GSH-Px的活力,降低MDA含量,并能提高小鼠皮肤组织中Hyp的含量。     

鼻出血鼻内镜下胶原蛋白海绵鼻腔填塞的应用与临床研究

目的研究分析鼻内镜下胶原蛋白海绵在鼻出血的临床应用价值与临床意义。方法对传统的凡士林纱条和新型的胶原蛋白海绵止血材料进行鼻腔填塞止血的2组病例的临床资料进行回顾性分析,并对2种填塞方法在填塞期和抽取时患者的鼻腔疼痛和(或)头痛,控制出血的有效性和抽取填塞物的难易程度进行比较。结果胶原蛋白海绵在填塞期和抽取时患者鼻腔疼痛和(或)头痛比凡士林组轻(P<0.05),填塞期24h出血量差异无统计学意义(P>0.05)。结论胶原蛋白海绵具有压迫止血和防止鼻腔粘连双重功能,是一种理想的鼻腔填塞物。     

猪源纤维蛋白贴对大鼠肝脏及肝素化兔臀大肌创面的止血作用

目的:研究猪源纤维蛋白贴(DBT)对大鼠肝脏和肝素化兔臀大肌出血创面的止血效果。方法:48只大鼠和24只兔均随机分为假手术组、手术对照组(纱布止血)、医用胶原蛋白海绵组和DBT组,除假手术组外,其他各组动物分别建立大鼠肝脏出血模型或肝素化兔臀大肌静脉出血模型。记录各组动物止血时间、计算出血量;观察大鼠术后3、13周DBT降解、肝脏创面愈合及粘连情况;考察医用胶原蛋白海绵组和DBT组肝素化兔的再渗血率。结果:与假手术组比较,手术对照组动物的止血时间和出血量均显著升高(P<0.01);与手术对照组比较,DBT组和医用胶原蛋白海绵组动物的止血时间和出血量均显著减少(P<0.01)。术后3周和13周,各组大鼠创面均有不同程度粘连,但粘连评分比较差异无统计学意义(P>0.05);术后13周两给药组大鼠肝脏肝缘稍钝,但基本已恢复至术前状态,愈合状况良好;DBT能降解、吸收完全。DBT组兔的再渗血率为33.3%,医用胶原蛋白海绵组为66.7%。结论:DBT对脆性脏器和凝血功能障碍机体具有较好的止血效果,且能降解吸收;其作用效果与医用胶原蛋白海绵相当,且黏合强度略强于后者。     

人肌腱胶原蛋白的提取及凝胶制备

目的探索人肌腱胶原蛋白在组织工程皮肤及整形外科中的应用。方法从人肌腱提取酸溶性胶原,制备人肌腱胶原蛋白。结果所提取的胶原在pH5.8左右迅速形成凝胶。光镜下可见酸溶性胶原纤维呈长细丝状,交织成网状。纯度以羟脯氨酸质量分数为基准,分光光度法测定为95%。氨基酸成分主要为甘氨酸占35%,脯氨酸占10%。结论氨基酸分析表明,所提取的胶原为典型的Ⅰ型胶原蛋白,热稳定性测定Td为39～41℃。     

全溶性止血纤维的动物试验及临床应用研究

目的：研究全溶性止血纤维（FHF）的止血效果。方法：30只家兔，制作不同的损伤创面以及动静脉损伤模型，应用不同材料进行止血，比较止血时间和出血量，观察不同材料肝素化前后的止血效果；214例外科手术患者随机分两组，107例为实验组应用FHF，对照组分为明胶海绵组和凡士林油纱组，比较止血时间和止血效果。结果：FHF优于明胶海绵的止血效果（P〈0．05）；动物肝素化前后FHF止血效果无明显差异，但与明胶海绵组相比有显著差异（P〈0．05）；临床实验结果可见，FHF在止血时间上明显短于对照组。止血效果与对照组比较有明显差别（P〈0．05）。结论：全溶性止血纤维止血安全、效果可靠。     

庆大霉素止血胶原海绵的制备及体外释放度

目的：建立消炎止血胶原海绵的制备方法及体外释放度测定方法.方法：制备纯度较高的胶原蛋白溶液,与硫酸庆大霉素和壳聚糖溶液混合并用戊二醛聚合,制得具有缓释作用的胶原海绵,通过正交设计优化合理配方并制备消炎止血胶原海绵;采用生物测定法在0,24,48,72,96,120,144 h测定消炎止血胶原海绵的释放度,建立累积释放率-时间曲线.结果：确定了胶原海绵的合理配方并建立了消炎止血胶原海绵的制备方法;确定了体外释放度测定中合适的采样点,通过精密度和回收率的测定初步完成了体外释放度方法学的建立,通过批间重现性测定和批内均一性测定确定体外释放度的标准：24 h释放度为1 000～1 220 u·mL^-1,72 h释放度为280～340 u·mL^-1,120 h释放度为130～170 u·mL^-1.结论：建立了消炎止血胶原海绵的制备方法及体外释放度的质量控制方法.     

Extraction,characterization and biocompatibility evaluation of silver carp (Hypophthalmichthys molitrix) skin collagen

Collagen as a biomaterial is commonly obtained from terrestrial animals. However, nowadays, the use of collagen with terrestrial animals' sources due to possible transmission of infectious diseases and religious beliefs is restricted. This study was conducted to evaluate the physicochemical characterization, morphology, and biocompatibility of extracted collagen from silver carp fish skin by-product. Type I collagen was extracted from the silver carp fish skin by-product using acetic acid and pepsin enzyme. The results showed that the extraction yield of ASC and PSC was 43% and 59% (on a dry weight basis), respectively. The presence of two different α-chains confirmed the type I collagen structure for both collagens. Moreover, FTIR spectra investigation demonstrated the triple helical in ASC and PSC collagens. The PSC showed higher imino acids content than ASC. Hence, the fractional viscosity and DSC curves revealed higher denaturation temperature (30 °C), shrinkage temperature (81 °C), and melting temperature (209 °C) for PSC than ASC (29 °C, 77 °C, and 187 °C, respectively). Finally, observations of the microscopic and the cell viability evaluation confirmed biocompatibility and suitable structure to cell growth. Accordingly, the obtained collagen from silver carp fish skin can be a proper alternative for terrestrial animals’ collagen and a safe biomaterial for biomedical use.     

Action and biocompatibility of two collagen-based hemostatics in animal experiments

Efficacy and biocompatibility of two collagen hemostatic wound dressings (usual agent: Collastypt, fluid-stabilised agent: Lyostypt) were compared. In a prospective randomized study the local hemostatic effect (bleeding time and blood loss) of the two agents were investigated after liver trauma of 45 rats. Liver wounds which remained untreated served as controls. Both preparations significantly reduced bleeding. The fluid-stabilised agent was significantly superior to the second collagen agent of different manufacturing.     

Biological testing of hemostatic properties of various collagen preparations

In experiments on animals a method of studying electrostatic and hemostatic properties of collagens was worked out. Following hemostatic collagen sponges were used: Superstat (Interface Biomedical Laboratories Corporation, USA), Hemostatic Felt (B. Braun, BRD), collagen sponge and collagen foil (Central Tissue Bank of Transplantation Institute, Medical Academy, Warsaw). Biological experiments were performed on rabbits in 4 groups, depending of the implanted material. Basing on surgical macro- and microscopic evaluation it was found that collagen preparations used in the experiments shorten the duration of bleeding of damaged liver, and that biological reactivity of studied collagen preparations is proportional to the duration of their resorption time in tissues. The shortest resorption time gave the Superstat.     

Wound collagen deposition in rats: effects of an NO-NSAID and a selective COX-2 inhibitor

Selective cyclo-oxygenase (COX)-2 inhibitors and nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) exhibit reduced toxicity in the gastrointestinal tract, but may affect wound healing in other tissues. In this study, we have compared the effects of a selective COX-2 inhibitor (celecoxib), a nitric-oxide releasing derivative of naproxen (HCT-3012) and naproxen in a model of wound collagen deposition in the rat. Polyvinyl alcohol sponges were implanted subcutaneously in rats. The rats were treated daily for 5 days with the test drugs at equieffective anti-inflammatory doses. Naproxen (10 mg kg(-1)) significantly decreased (45%) collagen deposition at the wound site relative to the vehicle-treated control group. In contrast, HCT-3012 (14.5 mg kg(-1)) significantly increased (62%) collagen deposition, while celecoxib (10 mg kg(-1)) had no effect. Naproxen and HCT-3012 suppressed prostaglandin (PG) E(2) levels at the wound site and whole blood thromboxane synthesis to similar degrees. Celecoxib had no significant effect on wound fluid PGE(2) levels, but slightly reduced whole blood thromboxane synthesis (by 17%). COX-1 mRNA and protein were expressed in the wound exudate, the skin surrounding the wound and in normal skin. In contrast, COX-2 mRNA, but not protein, was expressed in wound and normal skin. These results demonstrate that HCT-3012 can significantly enhance collagen deposition at a wound site, despite inhibiting prostaglandin synthesis to the same extent as the parent drug. Nitric oxide-releasing NSAIDs may represent a safer alternative to standard NSAIDs for use as anti-inflammatory and analgesic agents by post-surgery patients.     

In vitro effects of Se-allylselenocysteine and Se-propylselenocysteine on cell growth, DNA integrity, and apoptosis

Two previously unevaluated selenium compounds, Se-allylselenocysteine (ASC) and Se-propylselenocysteine (PSC), have been shown recently to be active in the chemoprevention of experimentally induced mammary carcinogenesis. Other than their potential as chemopreventive agents, little is known about the pharmacological properties of these compounds. In this article, we report on the in vitro effects of ASC and PSC on cell growth inhibition, apoptosis, and the induction of DNA damage. The effects of ASC and PSC were examined in two mouse mammary epithelial cell lines derived from mammary hyperplasias. These cell lines, designated TM2H and TM12, have mutant or wild-type p53, respectively. It was observed that ASC but not PSC reduced, in a concentration- and time-dependent manner, the number of adherent cells in culture, and this suppressive effect was more prominent in TM12 than in TM2H cells. ASC was also found to induce alkaline-labile DNA damage and the oxidation of pyrimidines, and it also increased the rate of apoptosis. These changes were not seen by exposure to PSC or the sulfur analog of ASC. However, additional data obtained from the intact rat mammary gland suggest that the loss of DNA integrity induced by ASC might not be manifest in vivo at doses of ASC that inhibit carcinogenesis.     

纤维结合蛋白复方制剂在创伤修复模型上的实验研究

目的 探讨纤维结合蛋白(FN)复方制剂对创伤愈合的促进作用.方法建立小鼠全皮层创伤修复模型,随机分为生理盐水组、胶原蛋白溶液治疗组、HA溶液治疗组、FN溶液治疗组、FN复方制剂治疗组、阳性对照治疗组,通过观察创面愈合情况、测定创面愈合率,采取皮肤组织学标本进行HE染色及免疫组化染色,观察各组药物对创伤愈合的促进作用.结...     

Hemostatic effect of a novel sheeted fibrin adhesive agent, TO-193, on experimental incision models]

TO-193 (TachoComb) is a new fibrin adhesive agent that consists of a collagen sheet coated with fibrin glue. We compared the hemostatic effect of TO-193 in several experimental models with Beriplast P as a fibrin adhesive agent, Avitene and Novacol as a microfibrillar collagen hemostat, and a sponge-like collagen sheet as the constitutional parts of TO-193. In the in vitro bleeding model in which blood leaks out through cotton cloth, the pressure of TO-193 when blood leakage was observed was higher than those of Beriplast P, Avitene, Novacol and the collagen sheet, indicating that TO-193 possessed a strong adhesive effect on the bleeding surface. On the kidney resection surface, TO-193 showed a more potent adhesive effect than those of Beriplast P and the collagen sheet, suggesting that TO-193 has a potent hemostatic effect. In the liver resection, TO-193 significantly reduced the bleeding volume compared with that of Novacol in normal rats. Furthermore, the bleeding volume of TO-193 was about half that of Beriplast P and equivalent to that of Novacol even in anticoagulant-treated rats. From these data, it is expected that TO-193 would be a valuable hemostatic agent for clinical use since TO-193 possesses a potent adhesive ability on the bleeding resection surface and would certainly stop bleeding in both patients with normal coagulation and those with a low blood coagulation condition.     

The Effects of Cyclophosphamide and Azathioprine on Collagen in Skin and Granulation Tissue in Rats,and the Effects of Cyclophosphamide on Collagen in Human Skin

Abstract: Granulation tissue was produced in rats by subcutaneous implantation of cellulose sponges. The effect of daily intraperitoneal injections of cyclophosphamide or azathioprine on rat skin and granulation tissue was examined after 14 and 42 days. Skin biopsies from patients with glomerulonephritis were analyzed before and after 42 days of treatment with cyclophosphamide or placebo. In the rats, cytostatic treatment caused an increase in the dry weight of the skin, and azathioprine increased the dry weight and the protein content of the granulomas. The increase in the dry weight was accompanied by a decrease in water percentage. The alpha-amino nitrogen/OH-proline ratio in purified acid soluble collagen from skin and granulation tissue increased with the dose and duration of cytostatic treatment. No effect on the aldehyde content was observed. Cyclophosphamide caused a decrease in the alpha/beta ratio in acid soluble collagen from granulation tissue, but not in the collagen from the skin. Salt soluble collagen was increased in the skin after 14 days of cytostatic treatment, but remained unchanged in the granulation tissue. In human skin cyclophosphamide caused no statistically significant changes in the amount of salt soluble or total collagen. It is concluded, that daily treatment of rats with cyclophosphamide or azathioprine from 14 to 42 days seems to inhibit the catabolic processes in the skin and granulation tissue, to decrease the hydroxylation of proline in collagen, and to inhibit the intermolecular cross-linking in collagen.     

Effect of different doses of D-penicillamine and combined administration of D-penicillamine and methylprednisolone on collagen, glycosaminoglycans, DNA and RNA of granulation tissue, skin, bone and aorta in rats

D-penicillamine (D-pen) in doses of 20, 100 and 500 mg/kg/day or D-pen 100 mg/kg/day plus methylprednisolone (MP) 2.0 mg/kg/day was administered daily for 42 days to rats implanted with viscose-cellulose sponges. Operated, pairfed rats served as controls. D-pen increased the DNA content of granulation tissue, but had no effect on the amount of tissue produced. In contrast, high dose D-pen reduced the content of DNA and collagen in skin. A dose related inhibition of collagen crosslink formation occurred in all tissues, particularly in skin, as indicated by increased proportions of extractable collagen with increased alpha/beta chain ratio and aldehyde content. Moreover, low doses of D-pen increased the hydroxyproline/proline ratio of acid soluble skin collagen, presumably due to solubilization of type III collagen as demonstrated by SDS-polyacrylamide gel electrophoresis in the presence of 3.6 M urea. These changes were associated with increased skin fragility and edema plus excess elastin deposition in the aorta after high dose D-pen treatment. Low dose D-pen stimulated the 35S-sulphate uptake into the sulphated glycosaminoglycans (GAGs) of granulation tissue without altering their relative amounts, whereas high dose D-pen reduced the concentration of chondroitin-4/6-sulphate in skin. MP antagonized the solubilizing effect of D-pen on collagen, probably by inhibition of the collagen synthesis. In addition, MP inhibited the cell proliferation and GAG metabolism. Food restriction reduced the DNA content of granulation tissue. The inhibitory effect of D-pen on the formation of granuloma collagen crosslinks in the presence of unaltered rate of collagen biosynthesis may diminish the amount of fibrotic tissue due to increased degradability of crosslink deficient collagen. Simultaneous administration of MP may facilitate this effect by inhibiting the biosynthesis of collagen. However, long-term D-pen treatment seems to increase the susceptibility of normal tissues to mechanical injury.     

参蓝胶原栓的研制

目的：以胶原蛋白为主要原料,配以中药苦参、板蓝根,通过适当的方法,制备一种用于宫颈创面止血、消炎并能加速创面愈合的新型栓剂材料。方法：制备高纯度的胶原蛋白溶液,栓剂基质,加人中药苦参、板蓝根制成栓剂,然后进行理化性能检测及质量监测、毒理学试验和药效学试验,并对其止血、消炎性能进行评价。结果：该栓剂质量稳定,pH值为5．5,毒理学试验均为阴性。药效学动物试验显示一期愈合率为90％,平均愈合时间为5-6d。实验组与对照组相比较,止血和愈合的效果比较具差异有统计学意义（P〈0．01）。结论：参蓝胶原栓,既可止血、抗茵消炎,又有利于创面愈合、缩短愈合时间,具有临床应用价值。