方格星虫纤溶酶的分离纯化及其性质研究
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摘要
目的血栓性疾病一直是严重危害人类健康的常见多发性疾病,其发病率、致残率及死亡率都居于高位,它主要危害了心脏、脑、肺等组织器官的血管系统。血栓性疾病的治疗手段主要是溶栓治疗,溶栓药物虽然经历了三代的发展仍然存在一定的副作用。因此,寻找并开发更为安全高效的溶栓药物成为心脑血管性疾病治疗的重要内容。海洋生物因其体内所具有的特殊生理活性物质成为了新药开发的宝贵资源。本实验研究旨在从海洋无脊椎动物方格星虫(Sipunculus Nudus)体内分离纯化出一种新型溶栓物质,并对其主要理化性质及生物活性进行研究。
方法(1)采用DEAE Sepharose CL-6B阴离子交换柱层析、Sephadex G-75和Superdex 200凝胶过滤柱层析等多步色谱分离技术相结合分离纯化纤溶酶,用纤维蛋白平板法测定其纤溶活性,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定其相对分子质量。(2)采用纤维蛋白平板法测定其最适温度和最适pH;利用苯甲基磺酰氟(PMSF)、E-64、EDTA、β-ME和抑肽酶5种具有专一性的抑制剂作为其纤溶活性的抑制剂,确定其蛋白酶学分类;通过标准纤维蛋白平板法和加热纤维蛋白平板法,检测其是否具有激酶的活性。(3)采用凝块溶解法测定该酶的体外溶栓效果,溶血性实验,皮下出血性实验和急性毒性实验检测其生物安全性。
结果(1)方格星虫纤溶酶在方格星虫体内含量丰富,经过DEAE Sepharose CL-6B,Sephadex G-75和Superdex 200交换层析分离得到纤溶酶,在SDS-PAGE电泳图谱上显示为一条带,其相对分子质量为33.25kDa,纯化倍数为6.48,回收率为10.61%。(2)与蚓激酶作用方式类似,方格星虫纤溶酶具有直接水解纤维蛋白作用;同时通过对纤溶酶原的激活,从而间接水解纤维蛋白,具有激酶活性。(3)酶学相关研究表明:该酶在30-50℃温度范围内,具有优良的热稳定性,其中在40℃时酶活力最高;该酶活力在pH6.5-8.0时较稳定,最适反应pH为7.5;Ba2+离子对该酶活力有一定的抑制作用,其他离子如Mg2+、Ca2+、K+、Na+和Ag+不影响该酶的活性。(4)酶纤溶活性完全被苯甲基磺酰氟(PMSF)抑制,E-64、EDTA、β-ME和抑肽酶对其活性没有影响,是一种典型的丝氨酸蛋白酶。(5)溶血性实验、皮下出血性实验和急性毒性实验证明,该酶具有良好的生物安全性。
结论方格星虫纤溶酶具有显著的纤溶活性,且稳定性高,具有潜在的临床研究开发价值。
Objective Nowadays, thrombotic disease has become the most severe disease in the world. Thrombotic disease causes not only the high incidence but also high mortality and disability. It mainly damages the blood systems of heart, brain and lung. Thrombolytic therapy is the most important pathway in treatment of thrombotic disease. There are three generations of thrombolytic agents, but more or less side effects appear in each of them. It is important for medicinal experts to search for more safe and effective medicines to cure the patients. In the light of the unique advantage of marine lives, they are becoming more and more precious resources in the future. Sipunculus Nudus is a marine invertebrate animal living in the sediment of South China Sea. It is found that the coelomic fluid of Sipunculus Nudus dose not congeal after being separated for a long time. This suggests that it may contain anticoagulants or fibrinolytic substances. In this study, we try to purify fibrinolytic enzyme from a marine invertebrate-Sipunculus Nudus. In the sametime, we will study its thrombolytic effect and its safety.
Methods We used sephacryl gel filtration chromatography and ion exchange chromatography to separate and purify Sipunculus Nudus fibrinolytic enzyme. The purity and relative molecular weight of Sipunculus Nudus fibrinolytic enzyme were identified by SDS-PAGE. The fibrinolytic activity was determined by fibrin plate. Rabbits were used to measure thrombolytic effects.
Results (1) The fibrinolytic enzyme could be isolated and purified by using of DEAE Sepharose CL-6B, Sephadex G-75 and Superdex 200 respectively. SDS-PAGE showed that only a single band for the purified enzyme, and the molecular weight was estimated to be about 33.25 kDa.The recovery yield was 10.61%, and the specific activity increased by 6.48 folds over the extracted crude. (2) Sipunculus Nudus fibrinolytic enzyme could not only directly degrade fibrin but also indirectly hydrolyze fibrin through transforming plasminogen into plasmin. (3) The enzyme activity of Sipunculus Nudus fibrinolytic enzyme was stable in the temperature between 30℃and 50℃and the pH ranging from 6.5 to 8.0. The optimum temperature of enzyme was about 40℃and the optimum pH was 7.5 respectively. The enzyme activity was obviously inhibited by Ba2+, other metal ions including Mg2+、Ca2+、K+、Na+、Ag+ had no obvious effect on the enzyme activity. (4) PMSF was inhibitor of Sipunculus Nudus fibrinolytic enzyme. (5) The haemolytic test, hypodermic bleeding test and acute toxicity test were confirmed that Sipunculus Nudus fibrinolytic enzyme owned good bio-security.
Conclution Sipunculus Nudus fibrinolytic enzyme could obviously enhance fibrinolytic activity in these experiments. It was safe and effective modern antithrombotic pharmaceutics with potential clinical applied values.
方法(1)采用DEAE Sepharose CL-6B阴离子交换柱层析、Sephadex G-75和Superdex 200凝胶过滤柱层析等多步色谱分离技术相结合分离纯化纤溶酶,用纤维蛋白平板法测定其纤溶活性,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定其相对分子质量。(2)采用纤维蛋白平板法测定其最适温度和最适pH;利用苯甲基磺酰氟(PMSF)、E-64、EDTA、β-ME和抑肽酶5种具有专一性的抑制剂作为其纤溶活性的抑制剂,确定其蛋白酶学分类;通过标准纤维蛋白平板法和加热纤维蛋白平板法,检测其是否具有激酶的活性。(3)采用凝块溶解法测定该酶的体外溶栓效果,溶血性实验,皮下出血性实验和急性毒性实验检测其生物安全性。
结果(1)方格星虫纤溶酶在方格星虫体内含量丰富,经过DEAE Sepharose CL-6B,Sephadex G-75和Superdex 200交换层析分离得到纤溶酶,在SDS-PAGE电泳图谱上显示为一条带,其相对分子质量为33.25kDa,纯化倍数为6.48,回收率为10.61%。(2)与蚓激酶作用方式类似,方格星虫纤溶酶具有直接水解纤维蛋白作用;同时通过对纤溶酶原的激活,从而间接水解纤维蛋白,具有激酶活性。(3)酶学相关研究表明:该酶在30-50℃温度范围内,具有优良的热稳定性,其中在40℃时酶活力最高;该酶活力在pH6.5-8.0时较稳定,最适反应pH为7.5;Ba2+离子对该酶活力有一定的抑制作用,其他离子如Mg2+、Ca2+、K+、Na+和Ag+不影响该酶的活性。(4)酶纤溶活性完全被苯甲基磺酰氟(PMSF)抑制,E-64、EDTA、β-ME和抑肽酶对其活性没有影响,是一种典型的丝氨酸蛋白酶。(5)溶血性实验、皮下出血性实验和急性毒性实验证明,该酶具有良好的生物安全性。
结论方格星虫纤溶酶具有显著的纤溶活性,且稳定性高,具有潜在的临床研究开发价值。
Objective Nowadays, thrombotic disease has become the most severe disease in the world. Thrombotic disease causes not only the high incidence but also high mortality and disability. It mainly damages the blood systems of heart, brain and lung. Thrombolytic therapy is the most important pathway in treatment of thrombotic disease. There are three generations of thrombolytic agents, but more or less side effects appear in each of them. It is important for medicinal experts to search for more safe and effective medicines to cure the patients. In the light of the unique advantage of marine lives, they are becoming more and more precious resources in the future. Sipunculus Nudus is a marine invertebrate animal living in the sediment of South China Sea. It is found that the coelomic fluid of Sipunculus Nudus dose not congeal after being separated for a long time. This suggests that it may contain anticoagulants or fibrinolytic substances. In this study, we try to purify fibrinolytic enzyme from a marine invertebrate-Sipunculus Nudus. In the sametime, we will study its thrombolytic effect and its safety.
Methods We used sephacryl gel filtration chromatography and ion exchange chromatography to separate and purify Sipunculus Nudus fibrinolytic enzyme. The purity and relative molecular weight of Sipunculus Nudus fibrinolytic enzyme were identified by SDS-PAGE. The fibrinolytic activity was determined by fibrin plate. Rabbits were used to measure thrombolytic effects.
Results (1) The fibrinolytic enzyme could be isolated and purified by using of DEAE Sepharose CL-6B, Sephadex G-75 and Superdex 200 respectively. SDS-PAGE showed that only a single band for the purified enzyme, and the molecular weight was estimated to be about 33.25 kDa.The recovery yield was 10.61%, and the specific activity increased by 6.48 folds over the extracted crude. (2) Sipunculus Nudus fibrinolytic enzyme could not only directly degrade fibrin but also indirectly hydrolyze fibrin through transforming plasminogen into plasmin. (3) The enzyme activity of Sipunculus Nudus fibrinolytic enzyme was stable in the temperature between 30℃and 50℃and the pH ranging from 6.5 to 8.0. The optimum temperature of enzyme was about 40℃and the optimum pH was 7.5 respectively. The enzyme activity was obviously inhibited by Ba2+, other metal ions including Mg2+、Ca2+、K+、Na+、Ag+ had no obvious effect on the enzyme activity. (4) PMSF was inhibitor of Sipunculus Nudus fibrinolytic enzyme. (5) The haemolytic test, hypodermic bleeding test and acute toxicity test were confirmed that Sipunculus Nudus fibrinolytic enzyme owned good bio-security.
Conclution Sipunculus Nudus fibrinolytic enzyme could obviously enhance fibrinolytic activity in these experiments. It was safe and effective modern antithrombotic pharmaceutics with potential clinical applied values.
引文
[1] Yu IT, Li W, Wong TW. Effects of age, period and cohort on acute myocardial infarction mortality in Hong Kong [J]. Int J Cardiol. 2004, 97(1):63-68.
[2] Blann AD, Landray MJ, Lip GY.ABC of antithrombotic therapy: An overview of antithrombotic therapy [J]. BMJ. 2002, 325(7367):762-765.
[3] Turpie AG, Chin BS, Lip GY. ABC of antithrombotic therapy: Venous thromboembolism: treatment strategies [J]. BMJ. 2002, 325(7370):948-950.
[4] Sinnaeve P, Werf FVD. Thrombolytic therapy. State of the art [J]. Thromb Res. 2001, 103(1):571-579.
[5]焦炳华.海洋生命活性物质和海洋药物的研究与开发[J].第二军医大学学报, 2006, 27(1):5-7.
[6]李光壁,王昶,刘占广.海洋药物研究进展与展望[J].盐业与化工, 2009, 38(5):43-47.
[7]郭学武,李复雪.光裸星虫生殖周期的研究[J].热带海洋,1993, 12(2):69-75.
[8]王庆恒,杜晓东,黄洪艳等.湛江地区光裸星虫的生殖细胞发育和生殖周期[J].湛江海洋大学学报, 2005, 25(1):5-9.
[9]兰国宝,阎冰.方格星虫繁殖生物学研究[J].水产学报, 2002, 26(6):503-509.
[10]兰国宝,廖思明,阎冰.方格星虫胚胎与幼体发育的研究[J].热带海洋学报, 2003, 22(6):70-75.
[11] Meyer A, Lieb B. Respiratory proteins in Sipunculus nudus --Implications for phylogeny and evolution of the hemerythrin family [J]. CompBiochem Physiol B Biochem Mol Biol, 2010, 155(2):171-177.
[12] Brown CL, Michel C, DeVillez EJ. Properties and distribution of a chymotryptic-like enzyme from the sipunculan, Phascolopsis gouldii [J]. Canadian Journal of Zoology, 1982, 60(3):361-367.
[13] Michel C, DeVillez EJ. Cytological and histochemical observations on the secretions in the digestive tract of the sipunculan, Phascolopsis gouldii (Pourtales,1851) [J]. Cell Mol Biol. 1984, 30(3):257-267.
[14]黄玉良,黄群,黄哲元等.复方星虫口服液对小鼠免疫功能的影响[J].福建中医学院学报, 1998, 8(2):32-33.
[15]沈先荣,蒋定文,贾福星等.海洋星虫提取物的抗疲劳作用研究[J].中华航海医学与高气压医学杂志, 2003, 10(2):112-114.
[16]蒋定文,沈先荣,贾福星等.海洋星虫提取物的营养分析及免疫调节作用的初步观察[J].中国生化药物杂志, 2004, 25(2):96-97.
[17]沈先荣,蒋定文,贾福星等.方格星虫延缓衰老作用研究[J].中国海洋药物, 2004, 97(1):30-32.
[18] Cutler EB, Gibbs PE. A Phylogenetic Analysis of Higher Taxa in the Phylum Sipuncula [J]. Syst Biol, 1985, 34(2):162-173.
[19] Buchner T, Abele D, Portner HO. Oxyconformity in the intertidal worm Sipunculus nudus: the mitochondrial background and energetic consequences [J]. Comp Biochem Physiol B Biochem Mol Biol, 2001, 129(1):109-120.
[20] Rice ME, Reichardt HF. Larval development and metamorphosis of Siphonosoma cumanense(Keferstein,l867) [J]. American Zoologist. 1984, 24(1):47A.
[21] Rice ME. Factors Influencing Larval Metamorphosis in Golfingia misakiana (Sipuncula) [J]. Bulletin of Marine Science. 1986, 39(2):362-375.
[22]余瑞元,袁明秀,陈丽蓉主编.生物化学实验原理和方法[M].第二版.北京:北京大学出版社, 2005:232-235.
[23] Astrup T, Mullertz S. The fibrin plate method for estimating fibrinolytic activity [J]. Archives of Biochemistry and Biophysics. 1952, 40(2):346-351.
[24]丁贵平,蔡正森,王正刚.纳豆激酶的分离纯化及生化研究[J].氨基酸和生物资源, 2001, 23(3):13-16.
[25]谢秋玲,郭勇.纳豆激酶活性测定方法[J].广东药学, 2000, 10(6):8-10.
[26] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 [J]. Nature. 1970, 227(5259):680-685.
[27]韩进庭.苍耳子的药理作用与临床应用[J].现代医药卫生, 2008, 24(20):3067-3068.
[28]蒋仲青,单环刺螠纤溶酶的分离纯化及其药效学研究, [学位论文],中国海洋大学, 2009.
[29]郭金明,单环刺螠纤溶酶UFE的分离纯化和性质研究, [学位论文],中国海洋大学, 2008.
[30] Hua Y, Jiang B, Mine Y, et al. Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste [J]. J Agric Food Chem. 2008, 56(4):1451-1457.
[31]张云龙,沙蚕纤溶蛋白酶的生化与分子生物学研究, [学位论文],吉林大学, 2007.
[32] Atkins J, Glynn P. Membrane association of and critical residues in the catalytic domain of human neuropathy target esterase [J]. J Biol Chem. 2000, 275(32):24477-24483.
[33]朱正光,吴曙光.眼镜蛇毒纤维蛋白原溶解因子的纯化及理化性质的研究[J].中国生物化学与分子生物学报, 1999, 15(5):782-786.
[34]饶颖竹,陈蓉,阮倩玲等.纳豆激酶粗提液的体外溶栓抑菌实验[J].蛇志, 2004, 16(1):7-9.
[35]陈奇主编.中药药理研究方法学[M].第二版.北京:人民卫生出版社, 2006:168-169.
[36]李婷,尖吻蝮蛇毒中抗凝组分Acoagulatin的抗血栓活性研究, [学位论文],第一军医大学, 2004.
[37]覃公平主编.中国毒蛇学[M].第二版.广西:广西科学技术出版社, 1998:360.
[38]张萍萍,纤溶酶产生菌的发酵条件优化及酶学性质初探, [学位论文],河北大学, 2006.
[39]孟紫强主编.环境毒理学[M].北京:中国环境科学出版社, 2000:496-497.
[40]中国食品药品监督管理局.中药、天然药物急性毒性研究技术指导原则.2005.
[41]袁伯俊,廖明阳,李波主编.药物毒理学实验方法与技术[M].北京:化学工业出版社, 2007:200-211.
[42]王心如主编.毒理学基础[M].第四版.北京:人民卫生出版社, 2006:103-113.
[43]谢秀琼主编.中药新制剂开发与应用[M].第二版.北京:人民卫生出版社, 2000:540-545.
[1] Gotoh Y, Ishizuka Y, Matsuura T. Spheroid Formation and Expression of Liver-Specific Functions of Human Hepatocellular Carcinoma-Derived FLC-4 Cells Cultured in Lactose-Silk Fibroin Conjugate Sponges [J]. Biomacromolecules. 2011, 12(5):1532-1539.
[2]陈冀胜.海洋药物研究开发漫谈[J].中国医学科学院学报, 2001, 23(5):415-417.
[3]蒋庆锋,周有骏,王金政.海洋抗肿瘤药物研究进展[J].中国海洋药物, 2004, 23(6):58-61.
[4] Yovine A, Riofrio M, Blay JY, et al. Phase II study of ecteinascidin-743 in advanced pretreated soft tissue sarcoma patients[J]. J Clin Oncol. 2004, 22(5): 890-899.
[5]李青选,王雪雯.膜海鞘素B药理活性及临床研究进展[J].国外医学药学分册, 1989, 16(6) : 321-324.
[6] Margolin k, Longmate J, Synold TW. Dolastain 10 in metastatic melanoma: a phaseⅡand pharmokinetic trial of the California Cancer Consortium [J] . Invest New Drugs. 2001, 19(4):335.
[7] Bai RL, Pettit GR, Hamel E. Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites [J]. J Boil Chem. 1990, 265(28):17141-17149.
[8] Bai RL, Pettit GR, Hamel E. Structure-activity studies with chiral isomers and with segments of the antimitotic marine peptide dolastatin 10 [J]. Biochem Pharmacol. 1990, 40(8):1859-1864.
[9]赵锐,顾谦群,管华诗等.天然活性多肽的研究进展[J].天然产物研究与开发, 2000, 12(4):84-91.
[10] Kanzaki A, Takebayshi Y, Ren XQ, et al. Over Coming multidrug resistance in P-glycoprotein/MRD1-Overexpressing cell lines by ecteinascidin 743 [J]. Mol Cancer Ther. 2002, 1(14):1327-1334.
[11] Gajate C, An F, Mollinedo F. Differential cytostatic and apoptotic effects of ecteinascidin 743 in cancer cells [J]. J Biol Chem. 2002, 277(44):41580-41589.
[12] Fayette J, Coquard IR, Alberti L, et al. ET-743: a novel agent with activity in soft-tissue sarcomas [J]. Curr Opin Oncol. 2006, 18(4):347-353.
[13] Jayson GC, Crowther D, Prendiville J, et al. A phase I trial of bryostatin 1 in patients with advanced malignancy using a 24 hour intravenous infusion [J]. Br J Caner. 1995, 72(2):461-468.
[14] Scheid C, Prendiville J, Jayson G, et al. Immunomodulation in patients receiving intravenous Bryostatin 1 in a phase I clinical study: comparison with effects of Bryostatin 1 on lymphocyte function in vitro [J]. Canner Immunol Immunother. 1994, 39(4):223-230.
[15]易杨华,焦炳华,张燕霞等.现代海洋药物学[M].北京:科学出版社, 2006:501
[16]周慧萍,陈琼华.紫菜多糖对实验性血栓和心肌收缩力的影响[J].中国药科大学学报, 1991, 22(1):50-52.
[17]彭波,项辉,赵金华等.褐藻多糖硫酸酯影响血小板聚集及血栓形成[J].中山大学学报论丛, 2002, 22(1):236-240.
[18]方翼,陈騉,梁蓓蓓等.单次静滴D-聚甘酯注射液在中国健康人体的安全性[J].中国临床药理学杂志, 2004, 20(4):283-286.
[19] Kariya Y, Sakai T, Kaneko T, et al. Enhancement of t-PA-mediatedplasminogen activation by partially defucosylated glycosaminoglycans from the sea cucumber Stichopus japonicas [J]. J Biochem. 2002, 132(2):335-343.
[20] Miyazaki S, Ishii K, Nadai T. The use of chitin and chitosan as drug carries [J]. Chem Pharm Bull (Tokyo). 1981, 29(10):3067-3069.
[21]侯惠民.甲壳质——一种具有广泛应用价值的新材料[J].中国医药工业杂志, 1988, 19(7):328-333.
[22]谭天瑞,张晓明,张慧英等.β-环糊精与消炎痛复合物的研究[J].北京大学学报, 1988, 20(2):97-99.
[23] Beena MS, Chandy T, Sharma CP. Phenyl alanine, tryptophan immobilized chitosan beads as adsorbents for selective removal of immunoproteins [J]. J Biomater Appl. 1994, 8(4):385-403.
[24]魏新林,夏文水.甲壳低聚糖的生理活性研究进展[J].中国药理学通报, 2003, 19(6) : 614-617.
[25]卞雅娜,刘殿祥.壳聚糖的研究进展[J].职业与健康, 2002, 18(1):44-45.
[26] Benoit E. Mechanism of action of neurotoxins acting on theinactivation of voltagegated sodium channels [J]. C R Seances Soc Biol Fil. 1998, 192(3):409-436.
[27] Murata K, Legrard AM, Ishibashi Y, et al. structures and Gnfigurations of eiguatoxin from the moray eel Cymnothorax javanicus and its likely precurson form the dinoflage Gambier discus toxious [J]. Jam Che Soc. 1990, 112, 8929-8931.
[28] Stake M, Murata M, Yasumoto T. The structure of CTX3C,a ciguatoxin congener isolated from Cultured Gambier discus toxicus [J]. Tetrahedron Letters. 1993, 34(12):1975-1978.
[29] Stake M, Ishibashi Y, Legrand AM, et al. Isolation and structure of ciguatoxin-4A,a new ciguatoxin prexursor,from culture of dinonflagellate Gambierdiscus toxicus and parrotfish Scarus gibbus [J]. Biosici Biotechnol Biochem. 1996, 60(12): 2103-2105.
[30]林永成,周世宁.海洋微生物及其代谢产物[M].北京:化学工业出版社, 2003:294-352.
[31] Gusovsky F, Daly JW. Maitotoxin: a unique pharmacological tool for research on calcium-dependent mechanisms [J]. Biochem Pharmacol. 1990, 39(11):1633-1639.
[32] Takahashi M, Ohizurai Y, Yasumoto T. Maitotoxin. A calcium activator Candidate [J]. J Biol Chem. 1982, 257():7287-7289.
[33] Manabu Nttldna. Koyanagi LM. Scheuet Paul J [J]. Toxicon, 1984, 22(2): 169.
[34]宋杰军,毛庆武.海洋生物毒素学[M].北京:北京科学技术出版社,1996:72-79.
[35] England LJ, Imperial J, Jacobsen R, et al. Inactivation of a serotonin-gated ion channel by a polypeptide toxin from marine snails [J]. Science. 1998, 281(5376):575-578.
[36] McIntosh JM, Jones RM. Cone venm-from accidental stings to deliberate injection. Toxicon. 2001, 39(10): 1447-1451.
[37] Jones RM, Bulaj G. Conotoxins-New Vistas for Peptide Therapeutics [J]. Curr Pharm Des. 2000, 6(12):1249-1285.
[38]易杨华,李玲,汤海峰.海洋药物研究进展[J].上海医药, 2003, 24(2):75-77.
[39] Cueto M, Jensen PR, Kauffman C, et al. Pestalone, a new antibioticproduced by a marine fungus in response to bacterial challenge [J]. J Nat Prod. 2001, 64(11):1444-1446.
[40] ParK CB, Lee JH, Park IK, et al. A novel antimicrobial peptide from the loach, Misgurnus anguillicaudatus [J]. FEBS Lett. 1997, 411(2-3):173-178.
[41] Asolkar RN, Maskey RP, Helmke E, et al. Chalcomycin B, a new macrolide antibiotic from the marine isolate Streptomyces sp. B7064 [J]. J Antibiot (Tokyo), 2002, 55(10):893-898.
[42] Barsby T, Kelly MT, Andersen RJ. Tupuseleiamides and basiliskamides, new acyldipeptides and antifungal polyketides produced in culture by a Bacillus laterosporus isolate obtained from a tropical marine habitat [J]. J Nat Prod. 2002, 65(10):1447-1451.
[43] Nagai K, Kamigiri K, Matsumoto H, et al. YM-202204, a new antifungal antibiotic produced by marine fungus phoma sp. [J]. J Antibiot (Tokyo), 2002, 55(12):1036-1041.
[44] Bai RL, Pettit GR, Hamel E. Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites [J]. J Boil Chem. 1990, 265(28):17141-17149.
[45] ParK CB, Lee JH, Park IK, et al. A novel antimicrobial peptide from the loach, Misgurnus anguillicaudatus [J]. FEBS Lett. 1997, 411(2-3):163-168.
[46] Oren Z, Shai Y. A class of highly potent antibacterial peptides derived from pardaxin, a pore-forming peptide isolated from Moses sole fish [J].Eur J Biochem. 1996, 237(1): 303-310.
[47] Nakamura T, Furunaka H, Miyata T, et al. Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachyplus tridentatus). Isolation and chemical structure [J]. J Biol Chem. 1988, 263(32):16709-16713.
[48] Lee IH, Zhao C, Cho Y, et al. Clavanins, alpha-helical antimicrobial peptides from tunicate hemocytes [J]. FEBS Lett. 1997, 400(2):158-162.
[49] Lee IH, Cho Y, Lehrer RI. Effects of pH and salinity on the antimicrobial peptides of clavanins [J].Infect Immun. 1997, 65(7):2898-2903.
[2] Blann AD, Landray MJ, Lip GY.ABC of antithrombotic therapy: An overview of antithrombotic therapy [J]. BMJ. 2002, 325(7367):762-765.
[3] Turpie AG, Chin BS, Lip GY. ABC of antithrombotic therapy: Venous thromboembolism: treatment strategies [J]. BMJ. 2002, 325(7370):948-950.
[4] Sinnaeve P, Werf FVD. Thrombolytic therapy. State of the art [J]. Thromb Res. 2001, 103(1):571-579.
[5]焦炳华.海洋生命活性物质和海洋药物的研究与开发[J].第二军医大学学报, 2006, 27(1):5-7.
[6]李光壁,王昶,刘占广.海洋药物研究进展与展望[J].盐业与化工, 2009, 38(5):43-47.
[7]郭学武,李复雪.光裸星虫生殖周期的研究[J].热带海洋,1993, 12(2):69-75.
[8]王庆恒,杜晓东,黄洪艳等.湛江地区光裸星虫的生殖细胞发育和生殖周期[J].湛江海洋大学学报, 2005, 25(1):5-9.
[9]兰国宝,阎冰.方格星虫繁殖生物学研究[J].水产学报, 2002, 26(6):503-509.
[10]兰国宝,廖思明,阎冰.方格星虫胚胎与幼体发育的研究[J].热带海洋学报, 2003, 22(6):70-75.
[11] Meyer A, Lieb B. Respiratory proteins in Sipunculus nudus --Implications for phylogeny and evolution of the hemerythrin family [J]. CompBiochem Physiol B Biochem Mol Biol, 2010, 155(2):171-177.
[12] Brown CL, Michel C, DeVillez EJ. Properties and distribution of a chymotryptic-like enzyme from the sipunculan, Phascolopsis gouldii [J]. Canadian Journal of Zoology, 1982, 60(3):361-367.
[13] Michel C, DeVillez EJ. Cytological and histochemical observations on the secretions in the digestive tract of the sipunculan, Phascolopsis gouldii (Pourtales,1851) [J]. Cell Mol Biol. 1984, 30(3):257-267.
[14]黄玉良,黄群,黄哲元等.复方星虫口服液对小鼠免疫功能的影响[J].福建中医学院学报, 1998, 8(2):32-33.
[15]沈先荣,蒋定文,贾福星等.海洋星虫提取物的抗疲劳作用研究[J].中华航海医学与高气压医学杂志, 2003, 10(2):112-114.
[16]蒋定文,沈先荣,贾福星等.海洋星虫提取物的营养分析及免疫调节作用的初步观察[J].中国生化药物杂志, 2004, 25(2):96-97.
[17]沈先荣,蒋定文,贾福星等.方格星虫延缓衰老作用研究[J].中国海洋药物, 2004, 97(1):30-32.
[18] Cutler EB, Gibbs PE. A Phylogenetic Analysis of Higher Taxa in the Phylum Sipuncula [J]. Syst Biol, 1985, 34(2):162-173.
[19] Buchner T, Abele D, Portner HO. Oxyconformity in the intertidal worm Sipunculus nudus: the mitochondrial background and energetic consequences [J]. Comp Biochem Physiol B Biochem Mol Biol, 2001, 129(1):109-120.
[20] Rice ME, Reichardt HF. Larval development and metamorphosis of Siphonosoma cumanense(Keferstein,l867) [J]. American Zoologist. 1984, 24(1):47A.
[21] Rice ME. Factors Influencing Larval Metamorphosis in Golfingia misakiana (Sipuncula) [J]. Bulletin of Marine Science. 1986, 39(2):362-375.
[22]余瑞元,袁明秀,陈丽蓉主编.生物化学实验原理和方法[M].第二版.北京:北京大学出版社, 2005:232-235.
[23] Astrup T, Mullertz S. The fibrin plate method for estimating fibrinolytic activity [J]. Archives of Biochemistry and Biophysics. 1952, 40(2):346-351.
[24]丁贵平,蔡正森,王正刚.纳豆激酶的分离纯化及生化研究[J].氨基酸和生物资源, 2001, 23(3):13-16.
[25]谢秋玲,郭勇.纳豆激酶活性测定方法[J].广东药学, 2000, 10(6):8-10.
[26] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 [J]. Nature. 1970, 227(5259):680-685.
[27]韩进庭.苍耳子的药理作用与临床应用[J].现代医药卫生, 2008, 24(20):3067-3068.
[28]蒋仲青,单环刺螠纤溶酶的分离纯化及其药效学研究, [学位论文],中国海洋大学, 2009.
[29]郭金明,单环刺螠纤溶酶UFE的分离纯化和性质研究, [学位论文],中国海洋大学, 2008.
[30] Hua Y, Jiang B, Mine Y, et al. Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste [J]. J Agric Food Chem. 2008, 56(4):1451-1457.
[31]张云龙,沙蚕纤溶蛋白酶的生化与分子生物学研究, [学位论文],吉林大学, 2007.
[32] Atkins J, Glynn P. Membrane association of and critical residues in the catalytic domain of human neuropathy target esterase [J]. J Biol Chem. 2000, 275(32):24477-24483.
[33]朱正光,吴曙光.眼镜蛇毒纤维蛋白原溶解因子的纯化及理化性质的研究[J].中国生物化学与分子生物学报, 1999, 15(5):782-786.
[34]饶颖竹,陈蓉,阮倩玲等.纳豆激酶粗提液的体外溶栓抑菌实验[J].蛇志, 2004, 16(1):7-9.
[35]陈奇主编.中药药理研究方法学[M].第二版.北京:人民卫生出版社, 2006:168-169.
[36]李婷,尖吻蝮蛇毒中抗凝组分Acoagulatin的抗血栓活性研究, [学位论文],第一军医大学, 2004.
[37]覃公平主编.中国毒蛇学[M].第二版.广西:广西科学技术出版社, 1998:360.
[38]张萍萍,纤溶酶产生菌的发酵条件优化及酶学性质初探, [学位论文],河北大学, 2006.
[39]孟紫强主编.环境毒理学[M].北京:中国环境科学出版社, 2000:496-497.
[40]中国食品药品监督管理局.中药、天然药物急性毒性研究技术指导原则.2005.
[41]袁伯俊,廖明阳,李波主编.药物毒理学实验方法与技术[M].北京:化学工业出版社, 2007:200-211.
[42]王心如主编.毒理学基础[M].第四版.北京:人民卫生出版社, 2006:103-113.
[43]谢秀琼主编.中药新制剂开发与应用[M].第二版.北京:人民卫生出版社, 2000:540-545.
[1] Gotoh Y, Ishizuka Y, Matsuura T. Spheroid Formation and Expression of Liver-Specific Functions of Human Hepatocellular Carcinoma-Derived FLC-4 Cells Cultured in Lactose-Silk Fibroin Conjugate Sponges [J]. Biomacromolecules. 2011, 12(5):1532-1539.
[2]陈冀胜.海洋药物研究开发漫谈[J].中国医学科学院学报, 2001, 23(5):415-417.
[3]蒋庆锋,周有骏,王金政.海洋抗肿瘤药物研究进展[J].中国海洋药物, 2004, 23(6):58-61.
[4] Yovine A, Riofrio M, Blay JY, et al. Phase II study of ecteinascidin-743 in advanced pretreated soft tissue sarcoma patients[J]. J Clin Oncol. 2004, 22(5): 890-899.
[5]李青选,王雪雯.膜海鞘素B药理活性及临床研究进展[J].国外医学药学分册, 1989, 16(6) : 321-324.
[6] Margolin k, Longmate J, Synold TW. Dolastain 10 in metastatic melanoma: a phaseⅡand pharmokinetic trial of the California Cancer Consortium [J] . Invest New Drugs. 2001, 19(4):335.
[7] Bai RL, Pettit GR, Hamel E. Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites [J]. J Boil Chem. 1990, 265(28):17141-17149.
[8] Bai RL, Pettit GR, Hamel E. Structure-activity studies with chiral isomers and with segments of the antimitotic marine peptide dolastatin 10 [J]. Biochem Pharmacol. 1990, 40(8):1859-1864.
[9]赵锐,顾谦群,管华诗等.天然活性多肽的研究进展[J].天然产物研究与开发, 2000, 12(4):84-91.
[10] Kanzaki A, Takebayshi Y, Ren XQ, et al. Over Coming multidrug resistance in P-glycoprotein/MRD1-Overexpressing cell lines by ecteinascidin 743 [J]. Mol Cancer Ther. 2002, 1(14):1327-1334.
[11] Gajate C, An F, Mollinedo F. Differential cytostatic and apoptotic effects of ecteinascidin 743 in cancer cells [J]. J Biol Chem. 2002, 277(44):41580-41589.
[12] Fayette J, Coquard IR, Alberti L, et al. ET-743: a novel agent with activity in soft-tissue sarcomas [J]. Curr Opin Oncol. 2006, 18(4):347-353.
[13] Jayson GC, Crowther D, Prendiville J, et al. A phase I trial of bryostatin 1 in patients with advanced malignancy using a 24 hour intravenous infusion [J]. Br J Caner. 1995, 72(2):461-468.
[14] Scheid C, Prendiville J, Jayson G, et al. Immunomodulation in patients receiving intravenous Bryostatin 1 in a phase I clinical study: comparison with effects of Bryostatin 1 on lymphocyte function in vitro [J]. Canner Immunol Immunother. 1994, 39(4):223-230.
[15]易杨华,焦炳华,张燕霞等.现代海洋药物学[M].北京:科学出版社, 2006:501
[16]周慧萍,陈琼华.紫菜多糖对实验性血栓和心肌收缩力的影响[J].中国药科大学学报, 1991, 22(1):50-52.
[17]彭波,项辉,赵金华等.褐藻多糖硫酸酯影响血小板聚集及血栓形成[J].中山大学学报论丛, 2002, 22(1):236-240.
[18]方翼,陈騉,梁蓓蓓等.单次静滴D-聚甘酯注射液在中国健康人体的安全性[J].中国临床药理学杂志, 2004, 20(4):283-286.
[19] Kariya Y, Sakai T, Kaneko T, et al. Enhancement of t-PA-mediatedplasminogen activation by partially defucosylated glycosaminoglycans from the sea cucumber Stichopus japonicas [J]. J Biochem. 2002, 132(2):335-343.
[20] Miyazaki S, Ishii K, Nadai T. The use of chitin and chitosan as drug carries [J]. Chem Pharm Bull (Tokyo). 1981, 29(10):3067-3069.
[21]侯惠民.甲壳质——一种具有广泛应用价值的新材料[J].中国医药工业杂志, 1988, 19(7):328-333.
[22]谭天瑞,张晓明,张慧英等.β-环糊精与消炎痛复合物的研究[J].北京大学学报, 1988, 20(2):97-99.
[23] Beena MS, Chandy T, Sharma CP. Phenyl alanine, tryptophan immobilized chitosan beads as adsorbents for selective removal of immunoproteins [J]. J Biomater Appl. 1994, 8(4):385-403.
[24]魏新林,夏文水.甲壳低聚糖的生理活性研究进展[J].中国药理学通报, 2003, 19(6) : 614-617.
[25]卞雅娜,刘殿祥.壳聚糖的研究进展[J].职业与健康, 2002, 18(1):44-45.
[26] Benoit E. Mechanism of action of neurotoxins acting on theinactivation of voltagegated sodium channels [J]. C R Seances Soc Biol Fil. 1998, 192(3):409-436.
[27] Murata K, Legrard AM, Ishibashi Y, et al. structures and Gnfigurations of eiguatoxin from the moray eel Cymnothorax javanicus and its likely precurson form the dinoflage Gambier discus toxious [J]. Jam Che Soc. 1990, 112, 8929-8931.
[28] Stake M, Murata M, Yasumoto T. The structure of CTX3C,a ciguatoxin congener isolated from Cultured Gambier discus toxicus [J]. Tetrahedron Letters. 1993, 34(12):1975-1978.
[29] Stake M, Ishibashi Y, Legrand AM, et al. Isolation and structure of ciguatoxin-4A,a new ciguatoxin prexursor,from culture of dinonflagellate Gambierdiscus toxicus and parrotfish Scarus gibbus [J]. Biosici Biotechnol Biochem. 1996, 60(12): 2103-2105.
[30]林永成,周世宁.海洋微生物及其代谢产物[M].北京:化学工业出版社, 2003:294-352.
[31] Gusovsky F, Daly JW. Maitotoxin: a unique pharmacological tool for research on calcium-dependent mechanisms [J]. Biochem Pharmacol. 1990, 39(11):1633-1639.
[32] Takahashi M, Ohizurai Y, Yasumoto T. Maitotoxin. A calcium activator Candidate [J]. J Biol Chem. 1982, 257():7287-7289.
[33] Manabu Nttldna. Koyanagi LM. Scheuet Paul J [J]. Toxicon, 1984, 22(2): 169.
[34]宋杰军,毛庆武.海洋生物毒素学[M].北京:北京科学技术出版社,1996:72-79.
[35] England LJ, Imperial J, Jacobsen R, et al. Inactivation of a serotonin-gated ion channel by a polypeptide toxin from marine snails [J]. Science. 1998, 281(5376):575-578.
[36] McIntosh JM, Jones RM. Cone venm-from accidental stings to deliberate injection. Toxicon. 2001, 39(10): 1447-1451.
[37] Jones RM, Bulaj G. Conotoxins-New Vistas for Peptide Therapeutics [J]. Curr Pharm Des. 2000, 6(12):1249-1285.
[38]易杨华,李玲,汤海峰.海洋药物研究进展[J].上海医药, 2003, 24(2):75-77.
[39] Cueto M, Jensen PR, Kauffman C, et al. Pestalone, a new antibioticproduced by a marine fungus in response to bacterial challenge [J]. J Nat Prod. 2001, 64(11):1444-1446.
[40] ParK CB, Lee JH, Park IK, et al. A novel antimicrobial peptide from the loach, Misgurnus anguillicaudatus [J]. FEBS Lett. 1997, 411(2-3):173-178.
[41] Asolkar RN, Maskey RP, Helmke E, et al. Chalcomycin B, a new macrolide antibiotic from the marine isolate Streptomyces sp. B7064 [J]. J Antibiot (Tokyo), 2002, 55(10):893-898.
[42] Barsby T, Kelly MT, Andersen RJ. Tupuseleiamides and basiliskamides, new acyldipeptides and antifungal polyketides produced in culture by a Bacillus laterosporus isolate obtained from a tropical marine habitat [J]. J Nat Prod. 2002, 65(10):1447-1451.
[43] Nagai K, Kamigiri K, Matsumoto H, et al. YM-202204, a new antifungal antibiotic produced by marine fungus phoma sp. [J]. J Antibiot (Tokyo), 2002, 55(12):1036-1041.
[44] Bai RL, Pettit GR, Hamel E. Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites [J]. J Boil Chem. 1990, 265(28):17141-17149.
[45] ParK CB, Lee JH, Park IK, et al. A novel antimicrobial peptide from the loach, Misgurnus anguillicaudatus [J]. FEBS Lett. 1997, 411(2-3):163-168.
[46] Oren Z, Shai Y. A class of highly potent antibacterial peptides derived from pardaxin, a pore-forming peptide isolated from Moses sole fish [J].Eur J Biochem. 1996, 237(1): 303-310.
[47] Nakamura T, Furunaka H, Miyata T, et al. Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachyplus tridentatus). Isolation and chemical structure [J]. J Biol Chem. 1988, 263(32):16709-16713.
[48] Lee IH, Zhao C, Cho Y, et al. Clavanins, alpha-helical antimicrobial peptides from tunicate hemocytes [J]. FEBS Lett. 1997, 400(2):158-162.
[49] Lee IH, Cho Y, Lehrer RI. Effects of pH and salinity on the antimicrobial peptides of clavanins [J].Infect Immun. 1997, 65(7):2898-2903.