培养的人脐静脉内皮细胞合成的蛋白聚糖

以[~(35)S]-Na_2SO_4为示踪物,观察培养的人脐静脉内皮细胞(EC)合成及分泌的蛋白聚糖(PG),经DEAE-Sephacel离子交换及Sepharose6B凝胶滤柱层析分析发现细胞层及培养液均含有三种PG单体,即硫酸乙酰肝素蛋白聚糖(HS-PG)、硫酸软骨素蛋白聚糖(CS-PG)及硫酸皮肤素蛋白聚糖(DS-PG)HS-PG又可分为大小两种,前者(HS-PG_L)位于V_o处,后者(HS-PG_s)Kd=0.53(sepharose6B);CS-PG/DS-PG分为三个峰,峰Ⅰ位于V_0处,峰Ⅱ、峰Ⅲ的Kd值分别为0.26及0.52(sepharose6B)。汇合前后细胞层及培养液中各种PG的含量不同。细胞层PG总量汇合前低于汇合后,无论是细胞层还是培养液汇合前HS-PG_L均低于汇合后,HS-PG_L与HS-PG_s比值亦为汇合前低于汇合后,而CS-PG/DS-PG含量则高于汇合后。汇合前后EC合成及分泌PG的差异与文献报道的EC损伤及正常者类似。     

人主动脉壁硫酸乙酰肝素蛋白聚糖对培养的人主动脉平滑机细胞合成蛋白聚糖

从人正常胸主动脉分离硫酸乙肝素蛋白聚糖,观察其对体外培养的人主动脉平滑肌细胞合成ＰＧ的影响,ＨＡＳＭＣ在不加或加ＨＳＰＧ的＾３５Ｓ－硫酸钠培养液中培养,以标记ＰＧ继之,培养液及细胞层的４ｍｏｌ／Ｌ盐酸胍提取液是ＰＧｓ经离子交换及凝胶过滤柱层析分离,发现加ＨＳＰＧ后,培养液中的ＨＳＰＧ,硫酸软骨素ＰＧ及硫酸皮肤素－硫酸软骨素ＰＧ（ＤＳＣＳＰＧ）均明显增高,而细胞层中仅ＨＳＰＧ和ＣＳＰＧ增高,且加ＨＳ     

培养的人主动脉平滑肌细胞合成的蛋白聚糖的生化分析

人胎儿主动脉平滑肌细胞(SMC_8)体外培养的第4(T_4)及第10(T_(10))代细胞在含[~(35)S]-硫酸钠的培养液中培养以标记蛋白聚糖(PG)。培养液及细胞层的4mol/L盐酸胍提取液中的PG_8用DEAE-Sephacel离子交换及凝胶过滤柱层析纯化。两代(T_4及T_(10))培养液中均含有一种分子较大及大小类似的硫酸软骨素-PG(CS-PG,在Sepharose CL-4B柱的排阻部位洗脱),其相对含量在T_4为20.8％,T_(10)为12.9％,另外尚均含有一种分子较小及大小类似的硫酸皮肤素-硫酸软骨素-PG(DS-CS-PG,Kd=0.27-0.33,Sepharose CL-4B),其相对含量在T_4为72.7％,T_(10)为81.5％。两代细胞层中除有一种与培养液中大小类似的CS-PG外(其相对含量在T_4为21.7％,T_(10)为10.2％),尚均含有一种分子较小(小于培养液中者)的DS-CS-PG(Kd=0.53,Sepharose CL-4B),其相对含量在T_4为57.4％,T_(10)为75.0％。另外,两代培养液及细胞层中均含有硫酸乙酰肝素-PG(HS-PG),在T_4及T_(10)培养液中分别为6.5％及5.6％;而在细胞层中则分别为20.9％及14.8％。T_(10)的培养液及细胞层中DS-CS-PG的[~(35)S]参入量均高于T_4者;而CS-PG及HS-PG则相反。T_(10)除有PG合成变化外尚有其他衰老迹象(如脂质堆积等),故SMC_8的老化可能与动脉粥样硬化病变形成有关。     

几种哺乳类动物胸主动脉中的蛋白聚糖

本文报道了北京(As高发)、广西南宁(As低发)两地区的人、兔(“易感”As)及狗(“不易感”As)胸主动脉中三种蛋白聚糖(CSPG,DSCSPG及HSPG)的含量。结果表明:a.广西南宁样品的总PGs,HSPG及DSCSPG含量均高于北京,尤以HSPG差异显著。b.兔、狗胸主动脉PG总量均低于人的。其中HSPG,CSPG含量及百分率以及兔的DSCSPG含量亦低于人的相应PG含量,而狗的DSCSPG含量与人的类似。c.南宁人及狗的DSCSPG含量及相对百分率虽高,但其DSCSPG中DS链所占相对百分率低于北京人和兔的。以上结果提示动脉壁PG质与量的差异可能与AS发病有关。     

人主动脉壁硫酸乙酰肝素蛋白聚糖对培养的人主动脉平滑机细胞合成蛋白聚糖的影响

从人正常胸主动脉分离硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）,观察其对体外培养的人主动脉平滑肌细胞（ＨＡＳＭＣ）合成ＰＧ的影响。ＨＡＳＭＣ在不加（对照）或加ＨＳＰＧ（１９μｇ醛酸／ｍｌ）的￣（３５）Ｓ－硫酸钠培养液中培养,以标记ＰＧ。继之,培养液及细胞层的４ｍｏｌ／Ｌ盐酸胍提取液中的ＰＧｓ经离子交换及凝胶过滤柱层析分离,发现加ＨＳＰＧ后,培养液中的ＨＳＰＧ,硫酸软骨素ＰＧ（ＣＳＰＧ）及硫酸皮肤素－硫酸软骨素ＰＧ（ＤＳＣＳＰＧ）均明显增高,而细胞层中仅ＨＳＰＧ和ＣＳＰＧ增高,且加ＨＳＰＧ后细胞层的ＤＳＣＳＰＧ分子大小有所不同,进一步分析ＤＳＣＳＰＧ中ＤＳ及ＣＳ含量发现加ＨＳＰＧ组ＨＡＳＭＣ细胞层中的ＤＳ％含量略低于对照组。结果提示ＨＳＰＧ可刺激ＨＡＳＭＣ的ＰＧ合成,其可能与血管壁修复及动脉壁脂质沉积有关。     

硫酸乙酰肝素蛋白聚糖对培养人脐静脉内皮细胞合成蛋白聚糖的…

以「＾３５Ｓ」－Ｎａ２ＳＯ４为示踪物,观察人正常主动脉中的硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对培养的第一代人脐静脉内皮细胞（ｈＵＶＥＣ）合成蛋白聚糖（ＰＧ）的影响,用解聚提取法及离子交换柱层析分离人主动脉ＨＳＰＧ,＾３５Ｓ－ＰＧｓ的混合物用离子交换及凝胶过滤柱层析法分离＾３５Ｓ－ＨＳＰＧ,＾３５Ｓ－硫酸软骨素－硫酸皮肤素ＰＧ（＾３５Ｓ－ＣＳＤＳＰＧ）及＾３５Ｓ－硫酸皮肤素ＰＧ（＾３５Ｓ－ＤＳＰＧ）,     

正常及损伤后修复的内皮细胞合成的蛋白聚糖

用自制尼龙刷将培养至汇合的人脐静脉内皮细胞刮伤后,造成规则的内皮细胞缺失区。继续培养可见,原有的内皮钿胞很快迁移到缺失区,并分裂增殖。约48小时新生的内皮细胞即将缺失区全部修复而形成新的汇合单层。以DEAE-Sephacel离子交换及Sepharose 6B凝胶过滤柱层析分析损伤后修复的内皮细胞合成的蛋白聚糖时发现所合成的蛋白聚糖总量减少;硫酸乙酰肝素蛋白聚糖合成相对减少、而硫酸软骨素及/或硫酸皮肤素蛋白聚糖合成相对增多。说明:伴随着内皮细胞的损伤后修复其蛋白聚糖的合成也有质和量的改变。     

灰树花蛋白聚糖的提取、分离及氨基酸组成分析

目的:研究灰树花(Grifola frondosa)蛋白聚糖的提取与分离方法及氨基酸组成.方法:采用碱提法提取蛋白聚糖,QSepharose FF离子交换层析进行纯化,SDS - PAGE电泳分离蛋白聚糖及结合蛋白,高效液相色谱法鉴定蛋白聚糖的纯度及分子量,并测定18种氨基酸的含量.结果:分离出蛋白聚糖GFP Ⅰ、GFPⅡ和GFPⅢ,糖含量分别为:43.69％、40.88％、28.33％,蛋白含量分别为:40.42％、34.65％、51.19％.其中GFPⅢ为均一成分,纯度93.61％,重均相对分子量(Mr) 150kDa,GFPⅢ结合蛋白分子量约为33.0kDa,为单一谱带.氨基酸组成分析结果显示:GFPⅢ的氨基酸总和达到66.60g/100g,其中8种必需氨基酸总量为26.14g/100g,4种呈味氨基酸总量为25.43g/100g,Asp、Glu、Leu、Ala和Lys含量较高.结论:分离出的蛋白聚糖为结构均一成分,纯度较高,可用于质量研究和生物学研究.     

硫酸乙酰肝素蛋白聚糖的功能机制研究进展

硫酸乙酰肝素蛋白聚糖是由核心蛋白和与之相连的硫酸乙酰肝素糖链组成,广泛分布于细胞膜与细胞外基质中。其中多配体蛋白聚糖（syndecan）和糖基磷脂酰肌醇锚定蛋白聚糖（glypican）存在与细胞膜上,而串珠蛋白聚糖（perlecan）和组合蛋白聚糖（agrin）表达在细胞外基质中。该类蛋白在生理与病理历程中,如发育、伤口愈合、肿瘤发生发展、感染、免疫应答等过程中担任重要作用,这些功能是其核心蛋白和糖链共同作用的结果。概述硫酸乙酰肝素蛋白聚糖的功能及其机制研究进展,同时强调其在作为药物靶标和临床诊断研究中的应用。     

聚集蛋白聚糖的硫酸化修饰与骨关节疾病

骨关节炎(osteoarthritis,OA)、大骨节病(Kaschin-Beck disease,KBD)等退行性骨关节病,均表现为软骨细胞外基质的过度降解和丢失。早期研究发现,O A和K B D病人软骨和关节滑液中糖醛酸含量增加,病人体内硫酸化程度降低。近年,糖生物学已成为生命科学的新前沿,研究提示糖复合物糖链修饰的改变与很多疾病的发生相伴随。蛋白聚糖(proteoglycan,PG)是一种在人和动物组织中分布广泛且重要的糖复合物,本文拟就聚集蛋白聚糖(aggrecan,A G)的结构及其代谢中硫酸化修饰与骨关节病研究进行综述,以期为骨关节病的深入研究和治疗提供新思路。     

Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor

Confluent cultures of human endothelial cells deposit into extracellular matrix (ECM) distinct heparan sulfate proteoglycans (HSPG) which modulate acidic fibroblast growth factor's (aFGF) ability to stimulate human endothelial cell mitogenic capacity. Extracellular matrix 35S-HSPG were isolated from cultures metabolically labelled with Na235SO4 by DEAE-Sepharose, Sepharose CL-4B, and aFGF-Affi-Gel 15 column chromatography and identified by resistance to chondroitinase ABC and sensitivity to nitrous acid. Fifty to sixty percent of the 35S-HSPG deposited into ECM do not bind aFGF. The bound 35S-HSGP (40-50% of the total counts applied) eluted from the aFGF-Affi-Gel column after the addition of buffer containing 2 M NaCl. aFGF-binding and aFGF-nonbinding 35S-HSPG were individually pooled and further purified by Sepharose CL-4B column chromatography. 35S-HSPG which bind aFGF, designated HSPGP, were 100-fold superior to heparin in augmenting the mitogenic efficacy of aFGF in sparse proliferating cultures. In contrast, however, 35S-HSPG, which did not bind aFGF, designated HSPG1, inhibited aFGF-stimulated proliferation in both sparse and subconfluent endothelial cell cultures. The majority of the biological activity of both aFGF-potentiating HSPGP and aFGF-inhibitory HSPG1 was contained in the glycosaminoglycan chains released by alkaline borohydride treatment of intact HSPGP or HSPG1, respectively. 3H-Core protein derived from HSPGP or HSPG1 contained only minor biological activity. The ability of heparitinase or heparinase (Flavobacterium heparinum) to abolish biological activity differed, depending upon the HSPG tested, also suggested that these are two distinct HSPGs.     

人主动脉硫酸乙酰肝素蛋白聚糖对培养人的脐静脉内皮细胞生长的影响

为探讨硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对内皮细胞生长的作用,用解聚提取及离子交换柱层析法分离出人主动脉ＨＳＰＧ,用倒置显微镜、细胞计数、及￣３Ｎ－ＴｄＲ参入观察其对培养的第一代人脐静脉内皮细胞（ｈＵＶＦＣ）生长的影响。结果发现：（１）倒置显微镜下观察,加入ＨＳＰＧ（１．７０μｇ已糖醛酸／ｍｌ）的ｈＵＶＥＣ生长密度高于对照组（未加ＨＳＰＧ）．（２）随着培养时间增加（２４,４８及７２ｈ）．根据细胞计数计算出同一剂量的ＨＳＰＧ（１７．０μｇ已糖醛酸／ｍｌ）对ｈＵＶＥＣ的促增殖％增高（分别为１４％,３０％及３７％）。（３）随着加入ＨＳＰＧ浓度的升高（４．３,８．５及１７．０μｇ已糖醛酸／ｍｌ．培养７２ｈ）．根据￣３Ｈ－ＴｄＲ参入计算出ＨＳＰＧ对ｈＵＶＥＣ的促增殖％亦增高（分别为４９％,７１％及９８％）。故人主动脉ＨＳＰＧ对培养的人脐静脉内皮细胞有促增殖作用。     

Serglycin is a major proteoglycan in polarized human endothelial cells and is implicated in the secretion of the chemokine GROalpha/CXCL1

Proteoglycan (PG) expression was studied in primary human umbilical vein endothelial cells (HUVEC). RT-PCR analyses showed that the expression of the PG serglycin core protein was much higher than that of the extracellular matrix PG decorin and the cell surface PG syndecan-1. PG biosynthesis was further studied by biosynthetic [(35)S]sulfate labeling of polarized HUVEC. Interestingly, a major part of (35)S-PGs was secreted to the apical medium. A large portion of these PGs was trypsin-resistant, a typical feature of serglycin. The trypsin-resistant PGs were mainly of the chondroitin/dermatan sulfate type but also contained a minor heparan sulfate component. Secreted serglycin was identified by immunoprecipitation as a PG with a core protein of ～30 kDa. Serglycin was furthermore shown to be present in perinuclear regions and in two distinct types of vesicles throughout the cytoplasm using immunocytochemistry. To search for possible serglycin partner molecules, HUVEC were stained for the chemokine growth-related oncogene α (GROα/CXCL1). Co-localization with serglycin could be demonstrated, although not in all vesicles. Serglycin did not show overt co-localization with tissue-type plasminogen activator-positive vesicles. When PG biosynthesis was abrogated using benzyl-β-D-xyloside, serglycin secretion was decreased, and the number of vesicles with co-localized serglycin and GROα was reduced. The level of GROα in the apical medium was also reduced after xyloside treatment. Together, these findings indicate that serglycin is a major PG in human endothelial cells, mainly secreted to the apical medium and implicated in chemokine secretion.     

Extracellular matrix derived from Trypanosoma cruzi infected endothelial cells directs phenotypic expression.

Infection of confluent human umbilical vein endothelial cells by the parasite Trypanosoma cruzi results in the appearance of an altered heparan sulfate proteoglycan (HSPG) isolated from the extracellular matrix of infected endothelial cells (ECMi). HSPG from ECMi differed from HSPG obtained from the extracellular matrix of uninfected endothelial cells (ECMu) by virtue of an 8-10-fold increase in sulfation and a different elution pattern using DEAE Sepharose chromatography. Analysis of the HSPG that binds to acidic fibroblast growth factor (aFGF) revealed that infection increased the proportion of HSPG that binds to aFGF by 35%. Heparitinase and alkaline borohydride treatment of aFGF-binding HSPG and chromatographic resolution on Sepharose CL4B column revealed an infection-associated 10-fold increase in sulfation of the GAG side chain with no significant change in the migration of the core protein. In addition, the aFGF binding HSPG isolated from ECMi demonstrated a markedly attenuated synergistic mitogenic activity with aFGF in a cell proliferation assay. All of the infection associated changes in HSPG could be demonstrated in HSPG obtained from uninfected endothelial cell cultures grown on ECMi. Hence, the ECMi is associated with signals capable of modulating the ECM associated metabolism of uninfected endothelial cells. This facility of ECMi was also shown to extend to patterns of Gs protein synthesis as revealed by Western blot analysis. The observation that the ECM produced by infected endothelial cells can direct the synthetic patterns of uninfected endothelial cells in a manner uniquely observed in infected endothelial cells suggests a plausible pathway by which infection of only a few cells can ultimately result in the coordinate responses of neighboring uninfected cells.     

Endothelial heparan sulfate is necessary but not sufficient for control of vascular smooth muscle cell growth

The state of the endothelial cell (EC) determines the nature of its control of vascular smooth muscle cell (vSMC) biology. Conditioned medium from postconfluent ECs inhibits vSMC proliferation, whereas subconfluent conditioned medium from the same ECs has a stimulatory effect. We and others have identified confluent endothelial cells' production of heparan sulfate proteoglycans (HSPG) as critical to vSMC growth control. The question that arises is whether the stimulation that is observed with subconfluent cells is from (1) aberrant HSPG production, (2) elaboration of noninhibitory species of HSPG, or (3) production of other factors, such as mitogens, which counteract the inhibitory HSPG to stimulate vSMCs. We studied the relative effects of conditioned medium produced by both subconfluent and postconfluent EC cultures on vSMC growth. Conditioned medium was fractionated into nonproteoglycan (non-PG) and proteoglycan (PG) components by anion-exchange chromatography. The PG fractionation profile and the antiproliferative activity of the HSPGs isolated from both subconfluent and postconfluent EC-conditioned media were similar. However, the HSPG fraction alone could not approach the inhibitory potential of unfractionated conditioned medium from postconfluent EC cultures. Non-PG proteins produced by the endothelial cultures had no effect on vSMC growth on their own. Yet, when they were mixed together with HSPG fractions, from either subconfluent or postconfluent EC cultures, the full growth effects were returned. Non-PG protein fractions from postconfluent cultures with HSPG fractions gave maximal inhibition of vSMC growth, whereas non-PG protein fractions from subconfluent EC cultures with HSPG fractions produced the maximal stimulation. Thus, whereas the net stimulatory or inhibitory effect on vSMC growth of EC-conditioned medium is density dependent, this effect does not result from a difference in the antiproliferative heparan sulfate component but rather from non-PG proteins that interact with the heparan sulfates.     

Heparan sulfate expression is affected by inflammatory stimuli in primary human endothelial cells

In diabetes the endothelium is either chronically or transiently exposed to hyperglycemic conditions. In addition, endothelial dysfunction in diabetes is related to changes in the inflammatory response and the turnover of extracellular matrix. This study was undertaken to study the effects of inflammatory stimuli on one particular matrix component, the heparan sulfate (HS) proteoglycans (PGs) synthesized by primary human umbilical cord vein endothelial cells (HUVEC). Such cells were cultured in vitro in 5 mM and 25 mM glucose. The latter concentration was used to mimic hyperglycemic conditions in short-term experiments. HUVEC were also cultured in the presence of the inflammatory agents tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), interleukin 1β (IL-1β) and transforming growth factor β (TGF-β). The cells were labeled with ³⁵S-sulfate and ³⁵S-PGs were recovered for further analyses. The major part of the ³⁵S-PGs was secreted to the medium, irrespective of type of stimuli. Secreted ³⁵S-PGs were therefore isolated and subjected to further analyses. TNF-α and IL-1α slightly increased the release of ³⁵S-PGs to the culture medium, whereas IL-1β treatment gave a significant increase. The different treatments neither changed the ratio of ³⁵S-HS and ³⁵S-chondroitin sulfate (CS) nor the macromolecular properties of the ³⁵S-PGs. However, the ³⁵S-HS chains were slightly increased in size after TNF-α treatment, and slightly decreased after TGF-β treatment, but not affected by the other treatments. Compositional analysis of labeled disaccharides showed changes in the amount of 6-O-sulfated glucosamine residues after treatment with TNF-α, IL-1α and IL-1β. Western immunoblotting showed that major HSPGs recovered from these cells were collagen XVIII, perlecan and agrin, and that secretion of these distinct PGs was increased after IL-1β stimulation. Hence, short term inflammatory stimuli increased the release of HSPGs in HUVEC and affected both the size and sulfation pattern of HS, depending on type of stimuli.     

Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties.

Human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) were labeled with 35SO(4)2- for 48 h. The membrane-associated proteoglycans were solubilized from these monolayers with detergent and purified by ion-exchange chromatography on Mono Q, incorporation in liposomes, and gel filtration. The liposome-intercalated proteoglycans were 125I-iodinated and treated with heparitinase before SDS-polyacrylamide gel electrophoresis. Radio-labeled proteins with apparent molecular masses of 130, 60, 46, 35, and 30 kDa (HAEC) and 180, 130, 62, 43, and 35 kDa (HUVEC) were detected by autoradiography. Further characterization by affinity chromatography on immobilized monoclonal antibodies and by Northern blot analysis provided evidence for the expression of syndecan, glypican, and fibroglycan in human endothelial cells. Most of the heparan sulfate which accumulated in the subendothelial matrix was implanted on a 400-kDa core protein. This protein was immunologically related to perlecan and bound to fibronectin. Binding studies on immobilized antithrombin III suggested that all membrane-associated heparan sulfate proteoglycan forms had the capacity to bind to antithrombin III but that high affinity binding was more typical for glypican. Most of the proteoglycans isolated from the extracellular matrix also bound only with low affinity to antithrombin III. These results imply that glypican may specifically contribute to the antithrombotic properties of the vascular wall.