猪凝血酶的分离纯化和部分性质研究

以猪血为材料,采用等电点沉淀和７２４树脂离子交换柱层析分离提纯,得凝血酶原,经ＣａＣｌ２激活后,通过ＤＥＡＥ－纤维素离子交换柱层析,获得比活为１２００ＩＵ／ｍｇ的凝血酶制剂,其收率为３２．１％。研究发现,ＮａＣｌ浓度对猪凝血酶活力有较大的影响,加１ｍｏｌ／ＬＮａＣｌ于凝血酶溶液中,室温（２５℃）放置３ｈ后,活力丧失５０％。猪凝血酶的最适温度和最适ｐＨ分别是３７℃和ｐＨ７．０。猪凝血酶在ｐＨ７．０、４０℃下放置２７ｈ,活力丧失８４％。通过ＳｅｐｈａｄｅｘＧ－１００凝胶层析,测出猪凝血酶的分子量为３０ｋｄ。     

猪凝血酶C端衍生肽抑菌活性和膜作用机制研究

猪凝血酶C末端富含正电荷和疏水性氨基酸残基,通过结构参数分析获得截短肽RD27,测定相应指标。探讨猪凝血酶C末端肽段抑菌活性及作用机制。结果表明,RD27对革兰氏阴性和阳性菌最小抑菌浓度为4~8μmol·L~(-1),对血细胞最小溶血浓度为45.9μmol·L~(-1),显示较强的抑菌活性和较低的溶血活性,治疗指数为10,大于蜂毒素。RD27在模拟膜环境中呈现α-螺旋结构,优先与模拟细菌脂质体作用,使E.coli UB1005膜产生去极化作用,并存在时间和剂量依赖效应。揭示其膜作用机制,拓宽抗菌肽设计方法,探究凝血与免疫系统联系。     

动物外用止血剂的制备

为提供外用止血剂用于兽医临床，本试验利用凝血酶原、凝血因子Xa、凝血因子V，采用正交试验设计确定了凝血酶原酶复合物各种成分的最佳浓度,从猪血中制备凝血酶，并根据兽医临床上的需要制成适合动物外用的止血剂型。该止血剂可以应用于兽医临床，是国内外兽医临床的新型外用止血剂。     

Changes in the Molecular Characteristics of Bovine and Marine Collagen in the Presence of Proteolytic Enzymes as a Stage Used in Scaffold Formation

Biopolymers, in particular collagen and fibrinogen, are the leading materials for use in tissue engineering. When developing technology for scaffold formation, it is important to understand the properties of the source materials as well as the mechanisms that determine the formation of the scaffold structures. Both factors influence the properties of scaffolds to a great extent. Our present work aimed to identify the features of the molecular characteristics of collagens of different species origin and the changes they undergo during the enzymatic hydrolysis used for the process of scaffold formation. For this study, we used the methods of gel-penetrating chromatography, dynamic light scattering, reading IR spectra, and scanning electron microscopy. It was found that cod collagen (CC) and bovine collagen (BC) have different initial molecular weight parameters, and that, during hydrolysis, the majority of either type of protein is hydrolyzed by the proteolytic enzymes within the first minute. The differently sourced collagen samples were also hydrolyzed with the formation of two low molecular fractions: Mw ~ 10 kDa and ~20 kDa. In the case of CC, the microstructure of the final scaffolds contained denser, closely spaced fibrillar areas, while the BC-sourced scaffolds had narrow, short fibrils composed of unbound fibers of hydrolyzed collagen in their structure.     

Monitoring unfractionated heparin therapy in dogs by measuring thrombin generation

Background: The calibrated automated thrombogram (CAT), an assay that permits measurement of thrombin generation in plasma, may be useful in studying hemostatic disorders and anticoagulant therapy in animals. Objectives: The aims of the study were to measure thrombin generation in healthy Beagle dogs and to evaluate the potential use of the CAT assay for monitoring therapy with unfractionated heparin (UFH). Methods: Individual platelet‐poor plasma samples and a plasma pool from 20 healthy adult Beagles were prepared. Serial UFH plasma dilutions were used to establish an in vitro heparin‐sensitivity curve. The pharmacodynamic effects of heparin in vivo were evaluated in Beagles using the CAT assay to measure thrombin generation with tissue factor at a concentration of 5 pM for initiation. Results: In healthy Beagles, the range of endogenous thrombin potential (ETP) was 238.7–414.0 nM/min (mean ± SD, 340.4 ± 63.1 nM/min). ETP intra‐assay and interassay variations were 7.1% and 12.9%, respectively. In vitro, a UFH concentration ≥0.4 U/mL resulted in total inhibition of thrombin generation. In vivo, the maximal effect of UFH on ETP was observed at 170 ± 36 minutes (range, 120–210 minutes) and resulted in a decrease in ETP of 38.5 ± 7.8% (range, 26.5–50.3%). In 210–420 minutes, ETP returned to baseline in 5 dogs. Conclusion: Our study demonstrates that thrombin generation can be measured in canine plasma and may be useful in assessing the degree of anticoagulation provided by UFH.     

半边旗中二萜类化合物5F对凝血酶诱导兔血小板聚集的影响

目的：研究半边旗中二萜类化合物５Ｆ（１１α－羟基－１５－氧－１６－烯－对映贝壳核烷－１９酸）对凝血酶诱导兔血小板聚集的影响。方法：血小板悬液与５Ｆ孵育１ｍｉｎ，然后用聚血酶（５０Ｕ／Ｌ）作用５ｍｉｎ，用血液凝集仪记录血小板聚集情况。结果：半边旗中二萜化合物５Ｆ对凝血酶诱导的兔血小反聚集有抑制作用。结论：半边旗中二萜类化合物５Ｆ具有抗血小板的作用。