注射用重组人干扰素α2b冻干制剂稳定性试验

本文通过对三批注射用重组人干扰素α２ｂ（ｒＩＦＮ－α２ｂ）冻干制剂在不同温度下,保存不同时间取样,对其外观、生物学活性、ｐＨ值、溶解状态及无菌试验等各项理化指标的观察试验,结果本品的冻干制剂在－２０℃和４－８℃的条件下保存４２个月,其上述各项检定指标无明显变化,均符合质检规程,即本文制品在此条件下稳定性良好,有效期可定为三年。同时说明该产品的生产工艺稳定。     

活菌制剂冻干保护剂的研究进展

冷冻干燥技术广泛应用于细菌性活疫苗生产和菌种保藏等方面。细菌细胞在冷冻干燥过程中会出现损伤,甚至死亡。通过添加适合的冻干保护剂可以最大程度减小细胞损伤,保持活菌制剂的活力和性能。就冻干过程对细菌的损伤机制、冻干保护剂的作用机理以及保护剂的筛选方法等方面进行了阐述,对活菌制剂保护剂的筛选有所启示。     

蛇毒类凝血酶试验方法研究

本文用蛇毒酶制剂和凝血酶对三种试验用稀释液和三种底物进行了凝固敏感性比较,然后选出最佳试验条件测定了８批蛇毒酶制品和三种蛇毒毒素。Ａ、Ｂ、Ｃ三中稀释液以Ｂ液凝固敏感性最高。０．２％牛纤原、０．２％人纤原和绵羊血浆三种底物以０．２％牛纤原敏感性最好。在没有牛纤原的情况下也可考虑选用人纤原,但牛纤原来源容易、成本低、不含人类传染病原体,应为首选底物。     

蛇毒中凝血酶样酶的研究

蛇毒中凝血酶样酶的研究韦世秀广西医科大学蛇毒研究所南宁５３００２１许多蝮亚科蛇毒中都含有一种氨基酸酯酶［１］,它催化纤维蛋白原分子特定部位Ａｒｇ－Ｇｌｙ肽键的裂解,释出血纤肽而转换为纤维蛋白。其作用与血浆凝血酶十分相似,因而称之为凝血酶样酶（Ｔｈｒｏ...     

人凝血因子Ⅷ制剂冻干工艺

优选了人凝血因子Ⅷ(FⅧ)制剂的冻干工艺。通过电阻法测定共晶点,再采用正交试验法,对预冻温度、主要干燥温度、主干燥时间、解析温度与真空压力等进行研究以优选冻干工艺,得到最佳的冻干工艺:-40℃预冻1.5 h;主要干燥温度从-40℃升温至-33℃,再从-33℃升温至0℃,总耗时36 h,真空压力为30 Pa;解析温度维持在35℃,真空压力为5 Pa,于终点测试压力无变化时结束。由于该工艺冻干出的人凝血因子Ⅷ制剂符合《中国药典》3部该项下的质量要求,经验证该冻干工艺能够应用于人凝血因子Ⅷ制剂的大规模生产。     

凝血酶抑制神经细胞突起生长的研究

本实验研究了凝血酶抑制CHP-100原始神经外胚叶瘤细胞神经分化的信号传导机制。凝血酶能抑制CHP-100细胞在无血清培养中神经突起的生长,这种作用与凝血酶激活细胞内磷酸肌醇/钙离子信号传导途径有关。凝血酶明显刺激Ins(1,4,5)P3的产生及细胞内游离钙离子浓度的升高。凝血酶的抑制剂水蛭素能抑制凝血酶引起的钙离子反应,并能拮抗凝血酶抑制CHP-100细胞神经突起生长的作用。结果提示,凝血酶信号传导系统可能在神经系统生长发育中具有重要调节作用。     

尖吻蝮(Agkistrodon acutus)蛇毒凝血酶样成分的研究——Ⅲ.蛇毒凝血酶止血效应的研究

将纯化得到的蛇毒凝血酶(TLE_3、TLE_4),经家兔体内实验表明,2—3凝血酶单位/kg体重剂量能显著地使家兔全血凝血时间缩短1/3—1/2。药后1小时即有促凝作用,以2—4小时凝血(止血)效应最强,12小时已消失。与Holleman, W. H.等自美洲矛头蝮蛇毒中得到的蛇毒凝血酶(Hemocoagulase)相似。经家兔及家犬实验性创伤止血实验表明,对创伤出血有止血作用。     

蛇毒类凝血酶的研究概况

自 1 936年 Klobusitzki和 Konig首次从美洲矛头蝮蛇 ( Bothrops jararaca)毒中获得部分纯化的类凝血酶以来 ,迄今已发现 30余种蛇毒中含有类凝血酶组份 ,并有 2 0余种先后得到分离和纯化。尤其是近几年来 ,有关蛇毒类凝血酶分子结构及酶学性质的研究取得了很大进展 ,部分已作为治疗药物而广泛应用于临床。兹就近几年来蛇毒类凝血酶的研究进展作一简要综述。1　蛇毒类凝血酶的分布　　以前认为蛇毒类凝血酶仅在于蝮亚科蛇毒中 ,蝰亚科中只有一种沙蝰 ( Vipera ammodytes)具有凝血酶样活性。随后不但从另一种蝰蛇加蓬咝蝰( Bitis gabonica)…     

碱性成纤维细胞生长因子制剂稳定性的研究

本研究通过MTT法检测bFGF的活性,研究了不同温度下四种制剂中bFGF的稳定性。结果得到,bFGF在2．0mol／L NaCl,0．05mol／L PB溶液(pH7.2)中,-20℃下贮藏6个月活性没有明显的下降;bFGF水剂贮藏在4℃下,bFGF活性随贮藏时间延长逐步下降,单位活性bFGF用量由0.4ng／mL下降为0．9ng／mL,室温下bFGF的活性丧失快于4℃下贮藏,3个月后bFGF单位活性用量由0．4ng／mL下降为1．0ng／mL。而乳霜剂和胶束剂中的bFGF在两种温度下贮藏一个月后,均检测不到对细胞生长的刺激作用。说明低温有利于制剂中bFGF的稳定,乳霜剂和胶束剂中的bFGF稳定性不好。     

长白蝮蛇类凝血酶基因大肠杆菌表达研究

从长白蝮蛇克隆的类凝血酶Ussurin Cdna被克隆进入pET-32α（＋）中与5′端与Thx-Tag和6xHis相连的T7启动子下,构建成大肠杆菌表达载体pET-32α（＋）-Ussurin;再转化感受态E.coli BL（21）DE3细胞,构成表达工程菌株;于IPTG诱导下表达,在细胞质中产生约占细胞质总蛋白10％的具有生物活性的Ussurin。     

Interaction of alpha 2-macroglobulin-bound thrombin with hirudin

The human thrombin bound to alpha 2-macroglobulin (alpha 2 M) in a 1:1 stoichiometry is still able to interact with one of its specific inhibitors, hirudin. The dissociation constant of the complex hirudin--alpha 2M-bound thrombin is 1 X 10(-7) M, whatever the mode of thrombin binding, covalent or non-covalent.     

Amplified electrochemical aptasensor for thrombin based on bio-barcode method

In the present study, an electrochemical aptasensor for highly sensitive detection of thrombin was developed based on bio-barcode amplification assay. For this proposed aptasensor, capture DNA aptamerI was immobilized on the Au electrode. The functional Au nanoparticles (DNA–AuNPs) are loaded with barcode binding DNA and aptamerII. Through the specific recognition for thrombin, a sandwich format of Au/aptamerI/thrombin/DNA–AuNPs was fabricated. After hybridization with the PbSNPs-labeled barcode DNA, the assembled sensor was obtained. The concentration of thrombin was monitored based on the concentration of lead ions dissolved through differential pulse anodic stripping voltammetric (DPASV). Under optimum conditions, a detection limit of 6.2 × 10⁻¹⁵ mol L⁻¹ (M) thrombin was achieved. In addition, the sensor exhibited excellent selectivity against other proteins.     

A colorimetric sandwich-type assay for sensitive thrombin detection based on enzyme-linked aptamer assay

A colorimetric sandwich-type assay based on enzyme-linked aptamer assay has been developed for the fast and sensitive detection of as low as 25 fM of thrombin with high linearity. Aptamer-immobilized glass was used to capture the target analyte, whereas a second aptamer, functionalized with horseradish peroxidase (HRP), was employed for the conventional 3,5,3′,5′-tetramethylbenzidine (TMB)-based colorimetric detection. Without the troublesome antibody requirement of the conventional enzyme-linked immunosorbent assay (ELISA), as low as 25 fM of thrombin could be rapidly and reproducibly detected. This assay has superior, or at least equal, recovery and accuracy to that of conventional antibody-based ELISA.     

A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin

A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH₃)₆]³⁺ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1–18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin.     

Inhibition of thrombin by synthetic hirudin peptides

To investigate the role of different regions of hirudin in the interaction with the proteinase thrombin, segments of hirudin containing 15-51 residues were synthesized. The C-terminal segment 40-65 inhibited the fibrinogen clotting activity of thrombin but not amidolysis of tosyl-Gly-Pro-Arg-p-nitroanilide. Central peptide 15–42 was insoluble at pH 7, but peptide 15-65 inhibited fibrinogen clotting and amidolysis to an equal extent. The N-terminal loop peptide 1-15 had no inhibitory activity and did not affect the potency of peptide 15-65. These data suggest that the central region inhibits catalysis.     

Heterocyclic core analogs of a direct thrombin inhibitor

Thrombin is a serine protease that plays a key role in blood clotting. Pyrrolidine 1 is a potent thrombin inhibitor discovered at Merck several years ago. Seven analogs (2–8) of 1 in which the pyrrolidine core was replaced with various heterocycles were prepared and evaluated for activity against thrombin, clotting factors VIIa, IXa, Xa, and XIIa, and trypsin. The thiomorpholine analog 6 was the most active, essentially matching the thrombin inhibitory activity of 1 with slightly improved selectivity over trypsin.     

Loop residues of thrombin-binding DNA aptamer impact G-quadruplex stability and thrombin binding

The thrombin-binding DNA aptamer (TBA) 5′-d(GGTTGGTGTGGTTGG)-3′ forms a G-quadruplex that is necessary for binding to the coagulation factor thrombin. The stability of the G-quadruplex of TBA when bound to thrombin and potassium ion (K⁺) were investigated for the wild-type oligonucleotide and for mutants in which thymine residues were substituted by adenine. In the presence of thrombin, G-quadruplexes formed by oligonucleotides in which the fourth or thirteenth residues were changed (T4A and T13A, respectively) were more unstable than that of wild-type, whereas T3A, T7A, T9A and T12A were more stable. The opposite effect was observed in the presence of 100 mM K⁺: the G-quadruplexes formed by T4A and T13A were more stable and T3A, T7A, T9A and T12A were more unstable than that of wild-type. Isothermal titration calorimetry measurements indicated that the binding constant of the interaction between T3A, T7A, T9A and T12A mutants and thrombin at 25 °C were close to that of wild-type, whereas T13A was significantly lower and T4A did not appear to bind to thrombin. Therefore, the stabilization of the G-quadruplex structure of TBA by thrombin appears to be due to an interaction between certain thymine nucleobases rather than to the quadruplex structure. The present study demonstrates that thrombin stabilizes the G-quadruplex via the interaction with residues in the loops but not via direct stabilization of G-quartets.     

Establishing the inhibitory effects of bradykinin on thrombin

Bradykinin, RPPGFSPFG, has been reported to be an inhibitor of thrombin's roles in blood clotting, platelet activation, and cellular permeability. The exact target, magnitude, and type of inhibition occurring are not well characterized. Based on the individual kinetic parameters calculated here, bradykinin is classified as a weak competitive inhibitor against hydrolysis of S-2238 and of a PAR4-like peptide. The K(m) values increased twofold in the presence of bradykinin, whereas the k(cat) values remained constant. The K(i) values ranged from 170 to 326 microM. Other biochemical studies indicated that bradykinin inhibits release of fibrinopeptide A from fibrinogen. Furthermore, bradykinin hindered the time required for fibrin clot formation. The weak inhibitions observed in vitro suggest that the direct effects of bradykinin on the thrombin active site become significant only at high concentrations, levels that may be difficult to achieve physiologically. Clearly, bradykinin can target thrombin but whether this direct interaction can be achieved in vivo and is sufficient to elicit a response without contributions from other cofactors requires further investigation.     

A paradoxical pro-apoptotic effect of thrombin on smooth muscle cells

Whereas thrombin (below 10 nM) is a potent mitogen, recent studies report that exposure to higher doses of thrombin could lead to apoptosis of neurons and tumor cells. Our results show that prolonged exposure (> or = 24 h) to thrombin (50-100 nM) exerts a pro-apoptotic effect on cultured vascular smooth muscle cells (VSMCs). This phenomenon depends on thrombin serine-protease activity but is independent of PAR-1 and -4 activation and subsequent signaling. The parallel occurrence of cell retraction and cleavage of fibronectin suggests that thrombin-induced apoptosis is consecutive to pericellular proteolysis. These data point to a new potential action of thrombin in the cardiovascular system.     

超声引导下股动脉假性动脉瘤内凝血酶注射加加压治疗的应用价值

目的：评价超声引导下股动脉假性动脉瘤瘤腔内注射凝血酶加加压治疗在治疗医源性股动脉假性动脉瘤中的应用价值。方法：在彩色多普勒超声引导下,采用20 G穿刺针经皮穿刺,对30例经股动脉介入治疗术后形成的股动脉假性动脉瘤患者行瘤腔内注射凝血酶封闭治疗同时行加压压迫治疗,凝血酶浓度为200 U/ml,总量均≤500 U,压迫治疗力量以病人能耐受,足背动脉搏动可触及为标准,加压时间为24小时。结果：30例患者均1次治疗成功,术中及术后无并发症发生,术后随访3个月无复发。结论：超声引导下股动脉假性动脉瘤瘤腔内注射凝血酶加加压治疗在治疗医源性股动脉假性动脉瘤中具有创伤小,操作简便,疗效确切的优点,可作为经股动脉介入治疗术后形成的假性动脉瘤的首选治疗方法。