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。     

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影响

以[35S」-Na2SO4为示踪物,观察人正常主动脉中的硫酸乙酸肝素蛋白聚糖（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）．结果发现实验组（加HSPG）与对照组（未加HSPG）相比,hU-VEC的35S-PGs总量（培养液+细胞层）无差别,但实验组培养液中35S-PGs总量升高、35S-DSPG、35S-CSDSPG及其相对百分含量均升高,而35S-HSPG及其百分含量降低．细胞层的35S-PGs,35S-HSPG及其相对百分含量降低,35S-DSPG及其相对百分含量升高,而CSDSPG未见差别．     

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）,     

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     

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。     

发酵法生产硫酸软骨素的研究进展

硫酸软骨素是一种典型的硫酸化糖胺聚糖,具有多种药物活性,广泛应用于药品、保健品及化妆品行业。硫酸软骨素是动物软骨中蛋白聚糖的主要成分和少数几种细菌的荚膜多糖,因此可利用动物提取法和发酵法进行生产。以下综述了硫酸软骨素的发酵生产及其合成机制的研究进展,并对其发展趋势进行了展望。     

不同缝合方式对大鼠角膜穿通伤后内皮细胞影响的研究

目的:通过手术缝合治疗大鼠角膜穿通伤,探索全层缝合、深板层缝合、及不缝合对角膜内皮细胞的影响.方法:建立大鼠角膜穿通伤模型,同一手术者对角膜切口进行全层、深板层、及不缝合操作.在裂隙灯下动态观察角膜创伤愈合情况;对不同时间点愈合角膜行内皮细胞台盼蓝-茜素红联合活细胞染色及HE染色,观察内皮细胞损伤、修复及白细胞浸润情况.结果:无论是全层缝合还是板层缝合以及不缝合组角膜内皮细胞均损伤明显.但从第1天观察至1月,三组损伤区面积大小无明显差别.结论:全层和深板层缝合及未缝合组可直接造成角膜内皮细胞受损,继发性炎症反应损伤后角膜内皮细胞的损害;伤口周围1.5 mm处角膜内皮几乎损失殆尽,内皮细胞受损可使角膜损伤区水肿迁延不愈,最终形成瘢痕愈合,所以角膜内皮损伤及最终愈合程度三组间无明显差异.     

人主动脉硫酸乙酰肝素蛋白聚糖对培养的人血管壁细胞生长的作用

通过培养的人主动脉平滑肌细胞（ｈＡＳＭＣ）及脐静脉内皮细胞（ｈＵＶＥＣ）,应用＾３Ｈ－ＴｄＲ参入、Ｎｏｒｔｈｅｒｎ ｂｌｏｔ分析、逆转录多聚酶链反应（ＲＴ－ＰＣＲ）、放射免疫分析（ＲＩＡ）、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对ｈＡＳＭＣ和ｈＵＶＥＣ ＤＮＡ合成的作用及对血小板源生长因子（ＰＧＤＦ）、ＰＧＤＦ受体、转化生长因子β（ＴＧＦ－β）、内皮素－１（ＥＴ－１）或     

超氧化物歧化酶对内皮细胞缺氧复氧损伤的防护作用

体外培养扔兔胸主动脉内皮细胞缺氧３０ｍｉｎ后复氧１０ｍｉｎ,可以发现缺氧后复氧可引起细胞乳酸脱氢酶释放量,细胞悬液丙二醛含量增加,谷胱甘肽过氧化酶活性降低,细胞合成释放一氧化氮减少,细胞内钙离子浓度明显升高;ＥＣ的这些损伤在缺氧期间即有表现,复氧后更为加剧。而在缺氧前预先加入终浓度为２００Ｕ／ｍｌ的超氧化物歧化酶可改善细胞的抗氧化能力,减轻缺氧复氧对ＥＣ的损伤。     

人主动脉硫酸乙酰肝系蛋白聚糖对培养人的脐静脉内皮细胞生长…

为探讨硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对内皮细胞生长的作用,用解聚提取及墩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＾３Ｈ－ＴｄＲ参入观察其对培养的第一代人脐静脉内皮细胞（ｈＵＶＥＣ）生长的影响,结果表明,（１）倒置显微镜下观察,加入ＨＳＰＧ（１．７０μｇ己糖醛酸／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     

Endothelial cell confluence regulates cyclooxygenase-2 and prostaglandin E2 production that modulate motility

Endothelial cells line the vasculature and, after mechanical denudation during invasive procedures or cellular loss from natural causes, migrate to reestablish a confluent monolayer. We find confluent monolayers of human umbilical vein endothelial cells were quiescent and expressed low levels of cyclooxygenase-2, but expressed cyclooxygenase-2 at levels comparable with cytokine-stimulated cells when present in a subconfluent culture. Mechanically wounding endothelial cell monolayers stimulated rapid cyclooxygenase-2 expression that increased with the level of wounding. Cyclooxygenase-2 re-expression occurred throughout the culture, suggesting signaling from cells proximal to the wound to distal cells. Media from wounded monolayers stimulated cyclooxygenase-2 expression in confluent monolayers, which correlated with the level of wounding of the donor monolayer. Wounded monolayers and cells in subconfluent cultures secreted enhanced levels of prostaglandin (PG) E(2) that depended on cyclooxygenase-2 activity, and PGE(2) stimulated cyclooxygenase-2 expression in confluent endothelial cell monolayers. Cells from subconfluent monolayers migrated through filters more readily than those from confluent monolayers, and the cyclooxygenase-2-selective inhibitor NS-398 suppressed migration. Adding PGE(2) to NS-398-treated cells augmented migration. Endothelial cells also migrated into mechanically denuded areas of confluent monolayers, and this too was suppressed by NS-398. We conclude that endothelial cells not in contact with neighboring cells express cyclooxygenase-2 that results in enhanced release of PGE(2), and that this autocrine and paracrine loop enhances endothelial cell migration to cover denuded areas of the endothelium.     

Cell density-dependent regulation of proteoglycan synthesis by transforming growth factor-beta(1) in cultured bovine aortic endothelial cells

The regulation of vascular endothelial cell behavior during angiogenesis and in disease by transforming growth factor-beta(1) (TGF-beta(1)) is complex, but it clearly involves growth factor-induced changes in extracellular matrix synthesis. Proteoglycans (PGs) synthesized by endothelial cells contribute to the formation of the vascular extracellular matrix and also influence cellular proliferation and migration. Since the effects of TGF-beta(1) on vascular smooth muscle cell growth are dependent on cell density, it is possible that TGF-beta(1) also directs different patterns of PG synthesis in endothelial cells at different cell densities. In the present study, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [(3)H]glucosamine, [(35)S]sulfate, or (35)S-labeled amino acids in the presence of TGF-beta(1). The labeled PGs were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chromatography. The glycosaminoglycan M(r) and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M(r) was analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain, heparitinase, or chondroitin ABC lyase. These experiments indicate that the effect of TGF-beta(1) on vascular endothelial cell PG synthesis is dependent on cell density. Specifically, TGF-beta(1) induced an accumulation of small chondroitin/dermatan sulfate PGs (CS/DSPGs) with core proteins of approximately 50 kDa in the medium of both dense and sparse cultures, but a cell layer-associated heparan sulfate PG with a core protein size of approximately 400 kDa accumulated only in dense cultures. Moreover, only in the dense cell cultures did TGF-beta(1) cause CS/DSPG hydrodynamic size to increase, which was due to the synthesis of CS/DSPGs with longer glycosaminoglycan chains. The heparan sulfate PG and CS/DSPG core proteins were identified as perlecan and biglycan, respectively, by Western blot analysis. The present data suggest that TGF-beta(1) promotes the synthesis of both perlecan and biglycan when endothelial cell density is high, whereas only biglycan synthesis is stimulated when the cell density is low. Furthermore, glycosaminoglycan chains are elongated only in biglycan synthesized by the cells at a high cell density.     

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.     

Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis

We have found that the spontaneous migration of bovine aortic endothelial cells from the edge of a denuded area in a confluent monolayer is dependent upon the release of endogenous basic fibroblast growth factor (bFGF). Cell movement is blocked by purified polyclonal rabbit IgG to bFGF as well as affinity purified anti-bFGF IgG and anti-bFGF F(ab')2 fragments. The inhibitory effect of the immunoglobulins is dependent upon antibody concentration, is reversible, is overcome by the addition of recombinant bFGF, and is removed by affinity chromatography of the antiserum through a column of bFGF-Sepharose. Cell movement is also reversibly inhibited by the addition of protamine sulfate and suramin; two agents reported to block bFGF binding to its receptor. The addition of recombinant bFGF to wounded monolayers accelerates the movement of cells into the denuded area. Transforming growth factor beta which has been shown to antagonize several other effects of bFGF also inhibits cell movement. The anti-bFGF IgG prevents the movement of bovine capillary endothelial cells, BHK-21, NIH 3T3, and human skin fibroblasts into a denuded area. Antibodies to bFGF, as well as suramin and protamine sulfate also suppress the basal levels of plasminogen activator and DNA synthesis in bovine aortic endothelial cells.     

The proteoglycan decorin is synthesized and secreted by differentiated myotubes

Proteoglycans (PGs) are important components of the skeletal muscle extracellular matrix (ECM). Skeletal muscles are composed of muscle fibers and mononucleated cells. The latter are known to synthesize and secrete several PGs. Rat skeletal muscle ECM contains a chondrotin/dermatan sulfate PG which was immunoprecipitated by antibodies against rat decorin. The synthesis and secretion of PGs by a mouse cell line was analyzed during in vitro differentiation. PGs were characterized by biochemical and immunological techniques including immunocytolocalization experiments. At least three different PGs are synthesized and secreted by differentiated myotubes: a 220 to 460 kDa heparan sulfate, a 250 to 310 kDa chondroitin/dermatan sulfate, and a 75 to 130 kDa chondroitin/dermatan sulfate. This latter PG was specifically immunoprecipitated with antibodies against rat fibroblast decorin. Indirect immunocytolocalization analysis revealed that decorin was localized inside the cells, with a strong reaction around the nuclei. During differentiation the relative proportions of some PGs changed. Thus, a decrease in the relative proportion of the heparan sulfate PG was observed, whereas a significant increase in the relative proportion of decorin was detected. No change in the large chondroitin/dermatan PG was seen during the differentiation process. The possible cell sources of decorin found in rat skeletal muscle ECM are discussed.     

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.     

Temporal regulation of hyaluronan and proteoglycan metabolism by human bone cells in vitro

Osteoblasts elaborate a dynamic extracellular matrix that is constructed and mineralized as bone is formed. This matrix is primarily composed of collagen, along with noncollagenous proteins which include glycoproteins and proteoglycans. After various times in culture, human bone cells were labeled with [35S]sulfate, [3H] leucine/proline, or [3H]glucosamine and the metabolism of hyaluronan and four distinct species of proteoglycans (PGs) was assayed in the medium, cell layer, and intracellular pools. These cells produce hyaluronan (Mr approximately 1,400,000; a chondroitin sulfate PG (CSPG), Mr approximately 600,000; a heparan sulfate PG (HSPG), Mr approximately 400,000; and two dermatan sulfate PGs with Mr approximately 270,000 (biglycan, PG I) and Mr approximately 135,000 (decorin, PG II) that distribute between the medium and cell layer. Two days following subculture, 12 h [35S]sulfate steady-state labeling yielded a composition of 24, 27, 31, and 18% for total CSPG, HSPG, biglycan, and decorin, respectively. While HSPG and decorin levels and distribution between medium and cell layer remained relatively constant during steady-state labeling at different times in culture, CSPG and biglycan levels increased dramatically at late stages of growth, and their distribution changed throughout culture. These results were independent of cell density, media depletion, and labeling pool effects. In contrast, hyaluronan synthesis was uncoupled from PG synthesis and apparently density-dependent. Pulse chase labeling at different stages of culture showed that the CSPG and decorin behaved as secretory PGs. Both HSPG and biglycan underwent catabolism, with HSPG possessing a t1/2 of 8 h and biglycan a t1/2 of 4 h. While the rate of HSPG turnover did not appreciably change between early and late culture, that of biglycan decreased. The mRNA for decorin was constant, while that of biglycan changed during culture. These results suggest that each PG possesses a distinct pattern of cellular and temporal distribution that may reflect specific stages in matrix formation and maturation.     

Non-enzymatic glycation of type I collagen diminishes collagen-proteoglycan binding and weakens cell adhesion

Non-enzymatic glycation of type I collagen occurs in aging and diabetes, and may affect collagen solubility, charge, polymerization, and intermolecular interactions. Proteoglycans(1) (PGs) bind type I collagen and are proposed to regulate fibril assembly, function, and cell-collagen interactions. Moreover, on the collagen fibril a keratan sulfate (KS) PG binding region overlaps with preferred collagen glycation sites. Thus, we examined the effect of collagen modified by simple glycation on PG-collagen interactions. By affinity coelectrophoresis (ACE), we found reduced affinities of heparin and KSPGs for glycated but not normal collagen, whereas the dermatan sulfate (DS)PGs decorin and biglycan bound similarly to both, and that the affinity of heparin for normal collagen decreased with increasing pH. Circular dichroism (CD) spectroscopy revealed normal and glycated collagens to assume triple helical conformations, but heparin addition caused precipitation and decreased triple helical content-effects that were more marked with glycated collagen. A spectrophotometric assay revealed slower polymerization of glycated collagen. However, ultrastructural analyses indicated that fibrils assembled from normal and glycated collagen exhibited normal periodicity, and had similar structures and comparable diameter distributions. B-cells expressing the cell surface heparan sulfate PG syndecan-1 adhered well to normal but not glycated collagen, and endothelial cell migration was delayed on glycated collagen. We speculate that glycation diminishes the electrostatic interactions between type I collagen and PGs, and may interfere with core protein-collagen associations for KSPGs but not DSPGs. Therefore in vivo, collagen glycation may weaken PG-collagen interactions, thereby disrupting matrix integrity and cell-collagen interactions, adhesion, and migration.     

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0-30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.