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首页> 外文会议>World biomaterials congress >Relationship of UDP-glucose dehydrogenase activity and in vitro hyaluronic acid release by human fibroblast-like synoviocytes
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Relationship of UDP-glucose dehydrogenase activity and in vitro hyaluronic acid release by human fibroblast-like synoviocytes

机译：人成纤维样滑膜细胞的UDP-葡萄糖脱氢酶活性与体外透明质酸释放的关系

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Introduction: Hyaluronic acid (HA) is an abundant glycosaminoglycan secreted by fibroblast-like synoviocytes (FLS) into the synovia! fluid. UDP-glucose dehydrogenase (UDPGD) is an enzyme expressed in FLS and a reported rate-limiting enzyme in HA synthesis'1!. FLS and macrophages form the synovial membrane and modulate together the composition of the synovial fluid, including HA levels and bioactive factors!2!!3'. To develop novel approaches to stimulate endogenous HA release into the synovial fluid, more knowledge is needed on the effect of cytokines on UDPGD activity. In this study, a novel in situ enzyme assay was developed to visualize UDPGD activity in primary FLS monolayer cultures, to test the hypothesis that anabolic, inflammatory and morphogenetic factors known to induce HA release also up-regulate UDPGD activity. We also tested the role of cell-cell contact in UDPGD activity using a new plastic disc method to generate controlled FLS aggregates in vitro. Methods: FLS cells were extracted from donated human synovial biopsies from consenting patients undergoing arthroscopic knee surgery (N=6) with mild synovitis and moderate knee pain, following approved protocols. UDPGD in situ enzyme staining of in vitro cell cultures was adapted from Pitsillides, used an extra cryoprotection step prior to staining, and unfixed rabbit synovium cryosections as controls. Confluent FLS cultures were incubated 24 hours in serum-free medium with and without IL-1β, PDGF-bb, TGF-β1, or 10% serum. HA release was measured by ELISA per DNA content. Cells were stained for in situ UDPGD activity in 8-well CultureSlide chambers. To create controlled cell aggregates, a fixed number of cells were attached to a specific area by capillary force using a sterile plastic disc. UDPGD stain was quantified by densitometry. Differences were calculated using Statistica (v10). Results: Specific UDPGD staining in confluent FLS cultures was obtained by a freeze-thaw step in a buffer that maintained cell morphology and eliminated false-positive oxidase stain. UDPGD activity and HA secretion were very low in serum-free medium. Despite donor-to-donor variation in UDPGD activity, staining results showed that UDPGD activity was intensified by serum, TGF-β1 and IL-1β and most strongly induced by PDGFbb. In vitro HA secretion in serum-free medium was increased 3-fold by serum and TGF-β1, and 6-fold by PDGFbb and IL-1β. The combination of TGF-β1 and IL-1β synergized to induce 30-fold greater HA release, with only minor UDPGD induction. Correlation analyses did not reveal a linear correlation between HA release and UDPGD activity. Instead, results showed that cell aggregation was the strongest inducer of UDPGD, with or without serum or cytokines. Cell aggregation was insufficient to induce HA secretion in serum-free medium. Discussion: Human FLS reproducibly up-regulated HA release in response to anabolic (PDGF-bb), catabolic (IL-1β) and morphogenetic (TFG-β1) factors. In these same conditions, we showed that UDPGD in situ enzyme activity is also induced, but not in direct proportion to the level of HA secreted in vitro. In addition, UDPGD activity was very low in the face of intense HA production that occurs under the synergic effect of catabolic and morphogenetic factors!4). It was previously reported that UDPGD activity is suppressed in highly inflamed knee synovial tissue. Data from this study suggest that the loss of UDPGD activity does not necessarily reflect loss of HA synthesis by FLS. The highest increase in UDPGD enzyme activity was observed in cell aggregates, which is consistent with the notion that cell-cell contact is a critical step prior to glycosaminoglycan deposition in the extracellular matrix. Conclusion: UDPGD in situ activity was variably induced by inflammatory cytokines and growth factors previously shown to induce FLS to secrete HA in vitro. These same factors are also known to be released by macrophages under inflammatory conditions or after phagocytosis of certain biomaterials. Intensification of UPDGD activity in cell aggregates may render cells competent to deposit glycosaminoglycan in the extracellular matrix.
机译：简介：透明质酸（HA）是一种丰富的糖胺聚糖，由成纤维样滑膜细胞（FLS）分泌到滑膜中！体液。 UDP-葡萄糖脱氢酶（UDPGD）是一种在FLS中表达的酶，是HA合成中报道的限速酶。 FLS和巨噬细胞形成滑膜，共同调节滑液的成分，包括HA水平和生物活性因子！2 !! 3'。为了开发刺激内源性HA释放到滑液中的新方法，需要更多有关细胞因子对UDPGD活性的影响的知识。在这项研究中，开发了一种新颖的原位酶测定法以可视化初级FLS单层培养物中的UDPGD活性，以检验以下假设：已知诱导HA释放的合成代谢，炎性和形态发生因子也上调UDPGD活性。我们还测试了细胞间接触在UDPGD活性中的作用，使用了一种新的塑料圆盘方法在体外产生受控的FLS聚集体。方法：按照批准的方案，从同意的接受轻度滑膜炎和中度膝痛的关节镜行膝关节手术（N = 6）的患者捐赠的人体滑膜活检样本中提取FLS细胞。体外细胞培养的UDPGD原位酶染色改编自Pitsillides，在染色前使用了额外的冷冻保护步骤，并将未固定的兔滑膜冷冻切片作为对照。将融合的FLS培养物在有无IL-1β，PDGF-bb，TGF-β1或10％血清的无血清培养基中孵育24小时。通过ELISA测定每个DNA含量的HA释放。在8孔CultureSlide室中对细胞进行原位UDPGD活性染色。为了产生受控的细胞聚集体，使用无菌塑料盘通过毛细作用力将固定数量的细胞附着到特定区域。 UDPGD染色通过光密度法定量。使用Statistica（v10）计算差异。结果：通过在保持细胞形态并消除假阳性氧化酶染色的缓冲液中进行冻融步骤，获得了融合FLS培养物中的特定UDPGD染色。在无血清培养基中，UDPGD活性和HA分泌非常低。尽管UDPGD活性的供体之间存在差异，染色结果显示，血清，TGF-β1和IL-1β增强了UDPGD活性，而PDGFbb最强地诱导了UDPGD活性。血清和TGF-β1使无血清培养基中的体外HA分泌增加了3倍，PDGFbb和IL-1β使HA分泌增加了6倍。 TGF-β1和IL-1β的组合可协同作用，诱导HA释放量增加30倍，而UDPGD的诱导作用却很小。相关分析未揭示HA释放与UDPGD活动之间的线性相关。相反，结果表明，无论有无血清或细胞因子，细胞聚集都是UDPGD的最强诱导剂。细胞聚集不足以诱导无血清培养基中的HA分泌。讨论：人类FLS可复制地上调HA释放，以响应合成代谢（PDGF-bb），分解代谢（IL-1β）和形态发生（TFG-β1）因子。在这些相同的条件下，我们表明UDPGD原位酶活性也被诱导，但并不与体外分泌的HA水平成正比。此外，在分解代谢和形态发生因素的协同作用下，发生大量HA时，UDPGD活性非常低！4）。以前有报道说，在高度发炎的膝滑膜组织中，UDPGD活性受到抑制。这项研究的数据表明，UDPGD活性的丧失并不一定反映FLS对HA合成的丧失。在细胞聚集体中观察到UDPGD酶活性的最高增加，这与以下想法一致：细胞-细胞接触是糖胺聚糖在细胞外基质中沉积之前的关键步骤。结论：UDPGD原位活性是由炎症细胞因子和生长因子可变地诱导的，以前已证明该因子可诱导FLS在体外分泌HA。还已知这些相同的因子在炎性条件下或某些生物材料的吞噬作用后由巨噬细胞释放。细胞聚集物中UPDGD活性的增强可能会使细胞具有在细胞外基质中沉积糖胺聚糖的能力。

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