Matrix glycosaminoglycans in the growth phase of fibroblasts: More of the story in wound healing
Matrix glycosaminoglycans in the growth phase of fibroblasts: More of the story in wound healing
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DOI：
10.1006/jsre.2000.5840
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发表时间：
2000-07-01
影响因子：
2.2
通讯作者：
Tromp, G
中科院分区：
文献类型：
--
作者：
Kosir, MA;Quinn, CCV;Tromp, G
背景资料。了解伤口愈合和加速愈合过程的方法包括了解影响肉芽组织合成的因素，肉芽组织在上皮化之前填充伤口，伤口早期愈合的一个重要阶段涉及成纤维细胞分泌糖胺聚糖(GAG)，在愈合过程中形成适合重塑的亲水基质。细胞外基质(ECM)中Gag结构和功能的复杂性在创伤愈合中的研究仍然很少。由于供体年龄、解剖部位或器官发育阶段的不同，目前尚无确定的皮肤伤口愈合模型。大鼠胚胎成纤维细胞(REF)作为研究成纤维细胞恶变的模型被用作伤口早期愈合的成纤维细胞模型，因为它们缺乏其他用于研究伤口早期愈合的成纤维细胞存在的基于年龄、部位和阶段的混杂变异。本研究的目的是鉴定和表征由REF-D、材料和方法合成的硫酸盐化GAG。将大鼠胚胎成纤维细胞(REF-D)培养在含血清的培养液中，并在生长过程中用S-35进行标记，以确定通常与蛋白多糖(PGs)相关的GAG链，并在含有蛋白酶抑制剂的情况下，用去污剂在pH 5.8的醋酸钠缓冲液中收集附着部位(富含ECM的)。用离子交换层析法收集硫酸盐分子，然后检测GAGS。用亚硝酸脱氨法测定硫酸乙酰肝素GAG，用软骨素酶测定硫酸软骨素/皮肤素GAG。单个GAG的比例是相对于分离的硫酸盐分子表示的。另外，从融合后的REF-D中提取RNA，用逆转录-聚合酶链式反应检测蛋白多糖的核心蛋白(核心蛋白核心蛋白聚糖、二聚糖、Syndecan-2和Perlecan)。GAG有两种主要构型：游离的GAG链(79.7%的硫化分子)和附着在蛋白多糖核心蛋白上的GAG(15.6%)。游离GAG链由硫酸软骨素(79.1%+/-3.5)和硫酸肝素(28.7%+/-2.1)组成。在较小的PG组中，硫酸乙酰肝素(94.8%+/-7.3)和硫酸软骨素(88.9%+/-3.2)链都附着在核心蛋白上。REF-D表达含有硫酸软骨素的PGs-Biglycan和Decorin的mRNA，另外还表达主要含有硫酸肝素链的Syndecan-2和Perlecan的PGs的mRNA。由次汇合的REF-D合成的大部分GAG链是硫酸软骨素，一小部分硫酸软骨素链作为PG的一部分与核心蛋白(例如，二聚糖、核心蛋白聚糖、Syndecan-2)结合。也存在硫酸乙酰肝素链，其中一小部分与核心蛋白有关(例如，PGS syndecan-2，perlecan)。更多的游离Gag链与周围分子形成弱相互作用，可能有助于在这一阶段移动和复制的成纤维细胞。因此，REF-D通过表达硫酸软骨素链和相关的PG而不受供体年龄、分期或解剖部位的影响，特别适合于研究伤口的早期愈合。(C)2000年学术出版社。
Background. Understanding wound healing and ways to accelerate the healing process includes understanding the factors that influence the synthesis of granulation tissue, which fills the wound before epithelialization, An important phase of early wound healing involves secretion of glycosaminoglycans (GAGs) by fibroblasts which form a hydrophilic matrix suitable for remodeling during healing. The complexity of GAG; structure and function in the extracellular matrix (ECM) remains poorly studied in wound healing. There is no established model for cutaneous wound healing due to variations in donor age, anatomic site, or stage of organ development. Rat embryo fibroblasts (REF) developed as a model to study malignant changes in fibroblasts were used as a model for fibroblasts in early wound healing because they lack the confounding variations based on age, site, and stage present in other fibroblasts used to study early wound healing. The purpose of this study was to identify and characterize the sulfated GAGs synthesized by REF-D,Materials and methods. Rat embryo fibroblasts (REF-D) were cultured in serum-based medium and radiolabeled during their growth phase with S-35 to identify the GAG chains usually associated with proteoglycans (PGs), The sites of attachment (ECM-rich) were collected with detergent in sodium acetate buffer, pH 5.8, in the presence of protease inhibitors. Sulfated molecules were collected by ion-exchange chromatography and then assayed for GAGs. Nitrous acid deamination was used to determine heparan sulfate GAGs, and chondroitinase was used for chondroitin/dermatan sulfate GAGs. The proportion of individual GAGs was expressed with respect to sulfated molecules isolated. In addition, RNA was isolated from subconfluent REF-D, and core proteins for proteoglycans (decorin, biglycan, syndecan-2, and perlecan) were assayed by reverse transcription polymerase chain reaction.Results. There were two major configurations of GAGs: free GAG chains (79.7% of sulfated molecules) and GAGs attached to the core protein of a proteoglycan (15.6%). The free GAG chains were composed of chondroitin sulfate (79.1% +/- 3.5) and heparan sulfate (28.7% +/- 2.1). In the smaller group of PGs, both heparan sulfate (94.8% +/- 7.3) and chondroitin sulfate (88.9% +/- 3.2) chains were attached to a core protein. REF-D expressed mRNA for biglycan and decorin, which are chondroitin sulfate-containing PGs, In addition, REF-D expressed mRNA for syndecan-2 and perlecan, which are PGrs that contain primarily heparan sulfate chains.Conclusions. A majority of GAG chains synthesized by subconfluent REF-D are chondroitin sulfate, A smaller proportion of chondroitin sulfate chains associate with a core protein as part of a PG (e.g., biglycan, decorin, syndecan-2). Heparan sulfate chains are also present, with a small proportion associated with a core protein (e.g., the PGs syndecan-2, perlecan). The greater presence of free GAG chains forming weak interactions with surrounding molecules may assist fibroblasts that are moving and replicating during this phase. Therefore, REF-D are particularly well suited to study early wound healing by their expression of chondroitin sulfate chains and associated PGs without the influence of donor age, stage, or anatomic site. (C) 2000 Academic Press.