低分子肝素的抗炎作用及机制

低分子肝素(low molecular weight heparin, LMWH)除作为抗凝血和抗血栓药在临床上广为应用外,近年来其抗炎活性也颇受重视.LMWH抗炎机制涉及炎症细胞、炎症因子和黏附分子等环节.目前对LMWH的抗炎机制研究还处在初级阶段,但是LMWH独特的性质使其有望成为有效且安全的新型抗炎药物.     

低分子量肝素对慢性阻塞性肺疾病大鼠的肺功能及气道炎性细胞的影响

目的:观察低分子量肝素(low molecular weight heparin,LMWH)对慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)大鼠肺功能及气道炎性细胞的影响。方法:将30只Wistar大鼠随机分为正常对照组、COPD模型组、LMWH干预组,每组10只。采用熏吸香烟法加气管内注入脂多糖的方法建立COPD模型,LMWH干预组于COPD模型开始建立时皮下注射150U/kg,每天一次,40天后检测各组大鼠肺功能及气道内炎性细胞的改变。结果:COPD模型组大鼠气道阻力及气道炎性细胞数均较正常组明显升高(P<0.05),以中性粒细胞为主;而皮下注射低分子量肝素治疗组气道阻力及气道炎性细胞数较COPD模型组均明显下降(P<0.05)。结论:低分子肝素可以减少COPD大鼠气道内炎性细胞数,降低气道阻力,有助于COPD病情的控制和转归。     

硫酸肝素蛋白多糖在疾病中的作用

硫酸肝素蛋白多糖广泛分布于动物组织的细胞膜和细胞外基质,对于机体发育和维持生理平衡至关重要.聚糖链硫酸肝素特有的分子结构使得这类大分子复合物具有多种生物功能,这些功能主要通过与蛋白质配体的结合实现.细胞表面的硫酸肝素蛋白多糖介导多种细胞活性因子与其受体的结合,参与信号转导的过程.硫酸肝素蛋白多糖也是细胞间质的重要组成部分,与胶原蛋白一起维持间质结构的稳定.肝素酶通过降解硫酸肝素从而调节细胞因子的活性和细胞间质的微环境.因此,揭示硫酸肝素的分子结构及其功能是生物学的一个重要研究方向.然而,由于硫酸肝素结构复杂,且不均一,使得这个领域的研究发展相对缓慢.不过,随着分析手段的提高和完善,国际上对于硫酸肝素结构与功能的报道迅速增加,同时国内对于硫酸肝素的研究也逐步受到重视.关于硫酸肝素的生理功能最近已有几篇比较全面的综述.此综述主要介绍硫酸肝素在病变中的作用,旨在探讨利用硫酸肝素和肝素酶作为靶标,研发预防和治疗这些疾病药物的可能性.     

大肠杆菌K5产肝素前体heparosan的研究进展

肝素是一种被广泛临床应用的抗凝血药物多糖。Heparosan是某些细菌荚膜中的GAG成分,其二糖骨架结构与脊椎动物中的肝素类似,可以作为肝素和硫酸乙酰肝素的生物合成前体。本文综述了肝素及肝素前体heparosan的功能与应用,heparosan在大肠杆菌K5中合成转运相关酶的研究,以及发酵法生产heparosan的研究进展,并对其应用前景进行了展望。     

肝素酶研究进展

微生物肝素酶是一类作用于肝素和类肝素的多糖裂解酶,在低分子肝素的制备以及肝素类分子的结构解析等领域具有十分重要的应用前景。本文将结合微生物基因组测序的最新进展,综述微生物肝素酶的来源,并对肝素酶的作用机理加以讨论;结合本实验室研究对肝素酶的重组表达及其在低分子肝素生产的应用最新进展作以综述,并对肝素酶其它潜在的应用作以讨论。     

硫酸乙酰肝素、肝素酶及其与肿瘤转移的关系

细胞外基质和基底膜的降解是癌细胞穿透组织屏障发生转移的重要步骤。硫酸乙酰肝素蛋白聚糖是细胞外基质和基底膜的组成成分,其多糖侧链可以被葡萄糖苷内切酶--肝素酶,特异性识别并切割,以破坏细胞外基质和基底膜的完整性,促进肿瘤转移。临床上肿瘤患者肝素酶高表达与肿瘤恶性程度和转移发生密切相关。深入了解硫酸乙酰肝素、肝素酶及它们与肿瘤转移相关的作用机制有助于我们寻找肿瘤治疗的新思路。本文将从硫酸乙酰肝素的合成调控、功能、肝素酶的转录和活性调节、肝素酶表达与肿瘤患者的临床特征,以及硫酸乙酰肝素、肝素酶与肿瘤转移的关系进行综述。     

肝素硫酸转移酶优化表达及其在动物源肝素硫酸化中的应用

肝素是一种重要的凝血药物,目前主要依赖于动物小肠粘膜的提取。动物源肝素含有的抗凝血活性五糖单位GlcNS6S-GlcA-GlcNS6S3S-Ido2S-GlcNS6S少,抗凝血活性低下。文中提出并验证了一种基于酶法催化动物源肝素,提高其硫酸化程度和抗凝血活性的方法。通过比较3种硫酸转移酶肝素-2-硫酸转移酶(Heparan sulfate-2-O-sulfotransferase,HS2ST)、肝素-6-硫酸转移酶(Heparan sulfate-6-O-sulfotransferase,HS6ST)、肝素-3-硫酸转移酶(Heparan sulfate-3-O-sulfotransferase,HS3ST)在重组大肠杆菌及重组毕赤酵母中表达,确定了毕赤酵母作为3种硫酸转移酶的表达宿主;进一步通过N端融合麦芽糖融合蛋白MBP和硫氧还蛋白Trx A,HS2ST和HS6ST的酶表达水平分别提高至(839?14) U/L和(792?23) U/L。通过3种硫酸转移酶HS2ST、HS6ST和HS3ST共同催化动物源肝素,其抗凝血活性由(76?2) IU/mg提高至(189?17) IU/mg。     

乙酰肝素酶的生物学特性的研究进展

乙酰肝素酶是切割哺乳动物细胞中硫酸肝素蛋白多糖侧链——硫酸乙酰肝素的内源性糖苷酶,是抗肿瘤转移的理想靶点。本就乙酰肝素酶的分子结构特点、亚细胞定位、活性调控机制、与肿瘤转移的关系、底物特异性和抑制剂开发等方面的研究进展进行了综述。     

乙酰肝素酶与胃癌发展的关系及其检测

乙酰肝素酶是目前发现的哺乳动物细胞中惟一能切割细胞外基质中硫酸乙酰肝素蛋白多糖侧链——硫酸乙酰肝素的一种葡萄糖醛酸内切酶,在胃癌侵袭转移中起重要作用。我们就乙酰肝素酶的分子结构特点、在胃癌侵袭转移中的作用机制及其检测等方面的研究进展进行综述。     

肝素C5异构酶的表达优化及分子改造

肝素和硫酸乙酰肝素是一类应用于临床抗凝血的糖胺聚糖。肝素葡萄糖醛酸C5异构酶(Heparosan-N-sulfate-glucuronate 5-epimerase,C5,EC 5.1.3.17) 是肝素和硫酸乙酰肝素合成过程中重要的修饰酶,催化N-硫酸化肝素前体 (N-sulfoheparosan) 的D-葡萄糖醛酸 (D-GlcA) 上5号位羧基翻转生成L-艾杜糖醛酸 (L-iduronic acid,L-IdoA)。文中以大肠杆菌Escherichia coli为宿主对斑马鱼来源的肝素葡萄糖醛酸C5异构酶基因Glce进行重组表达优化与分子改造。比较了3种不同的表达载体pET20b(+)、pET28a(+) 和pCold Ⅲ对C5表达的差异情况,其中以嗜冷启动型载体pCold Ⅲ表达酶活最高,达到(1 873.61±5.42) U/L。为了进一步提高C5的可溶表达量,在N端融合促溶标签SET2后,可溶蛋白表达量比对照提高了50%,酶活达到 (2 409.25±6.43) U/L。在此基础上,通过理性设计对底物结合口袋进行定点突变,获得最优突变体 (V153R) 的酶活和比酶活分别为 (5 804.32±5.63) U/L和(145.14±2.33) U/mg,是原始酶的2.41倍和2.28倍。肝素C5异构酶改造与表达优化为酶法催化合成肝素奠定了基础。     

The anti-cancer properties of heparin and its derivatives: a review and prospect

ABSTRACT

Heparin, including unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) and heparin derivatives, are commonly used in venous thromboembolism treatment and reportedly have beneficial effects on cancer survival. Heparin can affect the proliferation, adhesion, angiogenesis, migration and invasion of cancer cells via multiple mechanisms. The main mechanisms involve inhibition of heparanase, P-/L-selectin, angiogenesis, and interference with the CXCL12-CXCR4 axis. Here we summarize the current experimental evidence regarding the anti-cancer role of heparin and its derivatives, and conclude that there is evidence to support heparin’s role in inhibiting cancer progression, making it a promising anti-cancer agent.     

Effects of heparin and derivatives on podocytes: An in vitro functional and morphological evaluation

Unfractionated heparin (UFH) and low molecular heparin derivatives (LMWH) display numerous biological properties in addition to their anticoagulant effects. However, due to the physicochemical heterogeneity of these drugs, a better understanding concerning their effects on human cells is clearly needed. Considering that heparins are mainly excreted by the kidney, we focused our attention on the effect of UFH and LMWH on human podocytes by functional and morphological/phenotypic in vitro analyses. We demonstrated that these products differentially modulate the permeability of podocyte monolayer to albumin. The functional perturbations observed were correlated to significant cellular morphological and cytoskeletal changes, as well as a decrease in the expression of proteins involved in podocyte adherence to the extracellular matrix or intercellular interactions. This point confirms that UFH and the different LMWHs exert specific effects on podocyte permeability and underlines the need of in vitro tests to evaluate new biological nonanticoagulant properties of LMWH.     

肝素6位羧基修饰对抑制P?鄄选择素介导的A375细胞粘附活性的影响

已有的研究表明,肝素可以作为P-选择素的配体,显著抑制肿瘤转移过程中P-选择素介导的肿瘤细胞与血小板间的粘附.但是,肝素被P-选择素识别所必需的确切寡糖结构信息仍很缺乏.通过选择性化学修饰方法制备了2种低抗凝血肝素衍生物,即羧基还原肝素(CR-肝素)和羧基还原后再硫酸化肝素(SCR-肝素),系统地研究了它们对P-选择素介导的A375细胞粘附的抑制.研究结果表明,显著失去抗凝血活性的CR-肝素仍能有效地抑制P-选择素介导的A375细胞粘附,说明肝素的C6羧基并不是被P-选择素识别所必需的.而SCR-肝素所发生的C6羧基向羟甲硫酸酯基的转化却显著降低了抗粘附活性,说明P-选择素对肝素的识别并不只依赖于肝素的电荷密度.研究结果为深入阐明拮抗P-选择素介导的肿瘤细胞粘附的分子机制提供了有价值的实验基础.     

Selective cleavage of heparin using aqueous 2-hydroxypyridine: production of an aldose-terminating fragment with high anticoagulant activity

Unfractionated heparin (UFH) was partially depolymerized by heating at 115 degrees C with aqueous 2-hydroxypyridine. Compared to starting UFH, no significant loss of anticoagulant (anti-Xa) activity was observed. Products consisted of polysaccharide fragments and small quantities of ammonia, sulfate, and hexuronic acid. Fragments with aldose termini that reacted with [3H]NaBH4 (fragment A) were of relatively uniform size (6000 D) and increased as depolymerization time increased. Fragment A contained the anticoagulant activity, with 90-94% and 24-31% binding to Sepharose-thrombin and Sepharose-antithrombin, respectively. In contrast, a non-reducing fragment B that did not react with [3H]NaBH4 was more heterogeneous (6000-10,000 D) and did not have anticoagulant activity or Sepharose-antithrombin affinity. Given the polysaccharide 3H-incorporation, small release of monosaccharide products, and fragment A end-group analysis, thermolysis of UFH is likely limited to one site per molecule when protected by 2-hydroxypyridine. Thus, an anticoagulant fragment A is hydrolytically released from UFH leaving a variable-length fragment B complete with linkage region.     

肝素的抗炎作用与抗细胞粘附调节

肝素类药物具有抗凝以外的包括抗炎在内的多种生物学活性。炎症反应是多种因子、细胞参与的复杂的病生理过程,其物质基础是粘附分子介导的白细胞粘附及其粘附级联反应。近来研究证实肝素抗炎机制主要与抗细胞粘附调节有关,肝素通过竞争抑制L—、P—选择素与其配基sLe^X的结合,阻止白细胞粘附活化及调抑炎症级联反应,进而起到抗炎作用。对肝素抗炎机制的深入研究,将有助于进一步阐明抗粘附／抗炎的作用机制。     

低分子肝素与普通肝素对经皮冠脉介入手术的疗效和安全性的比较

目的:回顾性比较冠心病患者接受经皮冠脉介入(PCI)治疗时分别使用低分子量肝素(LMWH)和普通肝素(UFH)的疗效和安全性的差异。方法:选择本院2011-01至2012-12接受PCI术患者共200例,根据PCI使用肝素不同分为LMWH组(n=117),UFH组(n=83)。按常规方法,完成PCI术后,根据患者术中靶血管TIMI血流评价各组疗效,根据术中、后72h内出血/血肿、死亡、其他(心包填塞、胸痛、心源性休克、心脏破裂、室间隔穿孔、室速室颤、心脏骤停,阿斯发作、亚急性支架内血栓形成等)不良事件的发生率,比较各组安全性。结果:(1)各组患者临床基线资料差异无统计学意义(P0.05)。(2)各组患者PCI术中支架植入后靶血管血流改变存在统计学差异(P0.05),LMWH在疗效上优于UFH。(3)各组间死亡发生上无统计学差异(P0.05),在出血/血肿及其他(心包填塞、胸痛、心源性休克、心脏破裂、室间隔穿孔、室速室颤、心脏骤停,阿斯发作、亚急性支架内血栓形成等)的发生上存在统计学差异(P0.05),LMWH的不良反应少,安全性更高。结论:LMWH在PCI术中疗效更为显著,且较UFH不良反应少、安全性高,更适用于PCI术的抗凝治疗。     

Arginine functionalization of hydrogels for heparin binding—a supramolecular approach to developing a pro‐angiogenic biomaterial

Our aim was to synthesize a biomaterial that stimulates angiogenesis for tissue engineering applications by exploiting the ability of heparin to bind and release vascular endothelial growth factor (VEGF). The approach adopted involved modification of a hydrogel with positively charged peptides (oligolysine or oligoarginine) to achieve heparin binding. Precursor hydrogels were produced from copolymerization of N‐vinyl pyrolidone, diethylene glycol bis allyl carbonate and acrylic acid (PNDA) and functionalized after activation of the carboxylic acid groups with trilysine or triarginine peptides (PNDKKK and PNDRRR). Both hydrogels were shown to bind and release bioactive VEGF165 with arginine‐modified hydrogel outperforming the lysine‐modified hydrogel. Cytocompatibility of the hydrogels was confirmed in vitro with primary human dermal fibroblasts and human dermal microvascular endothelial cells (HUDMECs). Proliferation of HUDMECs was stimulated by triarginine‐functionalized hydrogels, and to a lesser extent by lysine functionalized hydrogels once loaded with heparin and VEGF. The data suggests that heparin‐binding hydrogels provide a promising approach to a pro‐angiogenic biomaterial. Biotechnol. Bioeng. 2013; 110: 296–317. © 2012 Wiley Periodicals, Inc.     

Measurement of the phospholipase activity of endothelial lipase in mouse plasma

Endothelial lipase (EL) is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans. Although this regulatory function is critically dependent on EL''s hydrolysis of HDL phospholipids, as yet there is no phospholipase assay specific for EL in plasma. We developed such an assay for the mouse enzyme using a commercially available phospholipid-like fluorescent substrate in combination with an EL neutralizing antibody. The specificity of the assay was established using EL knockout mice and its utility demonstrated by detection of an increase in plasma EL phospholipase activity following exposure of wild-type mice to lipopolysaccharide. The assay revealed that murine pre-heparin plasma does not contain measurable EL activity, indicating that the hydrolysis of HDL phospholipids by EL in vivo likely occurs on the cell surface.     

肝素在肿瘤治疗中的应用

肝素作为传统抗凝剂已众所周知。研究发现,肝素尚具有多种生物学活性,特别是抗肿瘤作用备受学者关注。尽管临床上并未将肝素疗法作为一种常规抗肿瘤手段,但是许多研究已经证明了肝素能够抑制肿瘤细胞的侵袭与转移。本文综述肝素治疗肿瘤的主要机制以及肝素结构修饰在抗癌方面的应用。     

The importance of Arg40 and 45 in the mitogenic activity and structural stability of basic fibroblast growth factor: Effects of acidic amino acid substitutions

High-affinity binding of basic fibroblast growth factor (bFGF) to the tyrosine kinase receptor requires cell-surface heparan sulfate proteoglycan or exogenous addition of heparin. The crystal structure of bFGF shows Arg40 and 45 on the surface opposite to the heparin-binding region, suggesting that these charged residues may be involved in the receptor binding. Therefore, these amino acids were mutated to aspartic acid separately or simultaneously, and also a simultaneous mutation to glutamic acid was introduced. These mutants displayed a mitogenic activity decreased greater than tenfold compared to the wild-type protein. Addition of heparin had no effect on the activity, while these mutants showed heparin-binding characteristics resembling those of the native sequence protein. The mutants exhibited decreased stability compared to the native sequence protein. Gradual changes in conformation were observed by circular dichroic and infrared spectroscopy. Heparin chromatography also showed the presence of denatured form for these mutants. However, in the presence of multivalent anions such as citrate, sucrose octasulfate, and heparin, the conformation of the mutants resembled that of the wild-type protein, as revealed by X-ray crystallography and circular dichroism spectra of the mutant with a Arg40 Asp substitution.Abbreviations FGF fibroblast growth factor - bFGF basic FGF - FBS fetal bovine serum - DMEM Dulbecco's Modified Eagles Medium - LMWH low-molecular-weight heparin - PBS phosphate-buffered saline - CD circular dichroism - FTIR Fourier transform infrared spectroscopy - MR molecular replacement - SIR single isomorphous replacement - EMTS ethylmercurithiosalicylate - SOS sucrose octasulfate