山羊颞下颌关节盘细胞透射电镜观察

目的 用透射电镜的方法对两只1月龄健康山羊四只颞下颌关节盘的细胞及胶原结构进行了观察.方法 切取山羊双侧颞下颌关节盘,经组织学处理后行透射电镜观察细胞和胶原超微结构特征.结果 颞下颌关节盘由不均分布的细胞和胶原纤维组成,细胞有成纤维细胞样细胞和软骨细胞样细胞两种,其中成纤维细胞样细胞较软骨细胞样细胞占优势.成纤维细胞样细胞表现有细长的突起,核大,梭形或不规则形,细胞器较少.软骨细胞样细胞胞核呈圆形或椭圆形,周围少有电子透射区,细胞膜不明显,胞突少见.胶原纤维由平行排列的有周期性横纹特征的胶原原纤维组成.结论 颞下颌关节盘细胞有成纤维细胞样和软骨细胞样细胞两种,前者占优势,胶原原纤维表现有周期性横纹特征.这为颞下颌关节盘组织工程研究中细胞来源及表型分析奠定了一定基础.     

选择性β肾上腺素受体激动剂对人滑膜细胞增殖能力的影响

目的：观察β-肾上腺素受体( AR)激动药对人成纤维样滑膜细胞( FLS)增殖的作用,探讨β-AR信号对人FLS功能的影响。方法组织块培养法培养人FLS;使用肿瘤坏死因子α( TNF-α)作为刺激剂,MTT法分别检测β1-AR选择性激动剂多巴酚丁胺和β2-AR选择性激动剂沙丁胺醇对人FLS增殖作用的影响,并计算增殖抑制率;使用沙丁胺醇+β2-AR选择性拮抗剂ICI 118551观察其对FLS增殖功能的影响;Western blot法检测人FLSβ-AR、G蛋白偶联受体激酶2(GRK2)和β抑制蛋白2(β-arrestin2)胞膜蛋白的表达水平。结果 TNF-α可以显著促进人FLS的增殖;β2-AR激动剂沙丁胺醇能明显抑制FLS的增殖,其拮抗剂ICI 118551能显著拮抗沙丁胺醇的作用;β1-AR选择性激动剂多巴酚丁胺对FLS的增殖无显著影响;TNF-α和沙丁胺醇均能显著降低β2-AR胞膜表达,上调 GRK2、β-arrestin2的胞膜表达。结论β2-AR信号的激活可以抑制FLS增殖;TNF-α和沙丁胺醇可以使GRK2、β-arrestin2的胞膜表达增加,β2-AR胸膜表达下降。     

Tumor necrosis factor-α inhibits chondrogenic differentiation of synovial fibroblasts through p38 mitogen activating protein kinase pathways

Abstract

We previously reported that synovial fibroblast-like cells (SFs) can be differentiated into chondrocytes through activin receptor-like kinase (ALK) 3 activation. The aim of this study was to clarify the effect and signaling pathways of tumor necrosis factor (TNF)-α on the chondrogenic differentiation of SFs. Primary SFs from patients with rheumatoid arthritis (RA) were treated with recombinant human bone morphogenetic protein-2 or transduced with a constitutively active mutant of the ALK3 gene (ALK3^CA) with or without TNF-α, and then cultured in pellets. Expression of chondrocyte-specific genes was analyzed by real-time polymerase chain reaction or by histological analysis. Inhibitors of mitogen-activating protein kinase (MAPK) pathways or adenovirus vectors carrying a dominant-negative mutant of the IκB kinase 2 gene (AxIKK2^DN) were used to analyze the signaling pathways of TNF-α. Expression of chondrocyte-specific genes was induced in SFs either by rhBMP-2 treatment or by ALK3^CA transduction, which was strongly suppressed by TNF-α treatment. TNF-α markedly increased the p38 MAPK pathways in SFs, and inhibition of p38 MAPK activation partially restored the inhibitory effect of TNF-α on the chondrogenic differentiation of SFs. Combination therapy BMP-2 and anti-TNF-α agents especially targeting p38 MAPK might be a good approach to stimulating neochondrogenesis in the damaged joints in RA.     

佐剂诱导关节炎大鼠模型的建立及成纤维样滑膜细胞的分离

目的建立类风湿性关节炎(RA)动物模型；从炎性关节滑膜中分离成纤维样滑膜细胞(FLS)。方法应用热灭活结核杆菌H37Ra菌株与矿物油混合制备改良的佐剂,尾根部皮内注射Lewis大鼠诱导关节炎；剪取成功诱导关节炎大鼠的病变踝关节,从中剥离滑膜组织,充分剪碎后采用胶原酶消化法分离成纤维样滑膜细胞。结果成功诱导了大鼠关节炎,发病率为100%,发病时间有规律,组织学表现与RA相似；成功在体外培养了FLS并对其进行了鉴定,掌握了其生长形态和特征。结论成功制备RA动物模型并获得FLS,为今后RA发病机制探索和药物评价提供了良好的体内动物模型和体外细胞模型。     

Network of telocytes in the temporomandibular joint disc of rats

The phenotypes of the temporomandibular joint (TMJ) disc cells range from fibroblasts to chondrocytes. There are relatively few reported studies using transmission electron microscopy (TEM) to determine the ultrastructural features of these cells. It was hypothesized that at least a subpopulation of TMJ stromal cells could be represented by the telocytes, cells with telopodes. In this regard a TEM study was performed on rat TMJ samples. Collagen-embedded networks were found built-up by cells with telopodes with subplasmalemmal caveolae, moderate content in matrix secretory organelles and well-represented intermediate filaments. Appositions of cell bodies were found. Prolongations of such cells were closely related to nerves and microvessels. Our study indicates that the TMJ disc attachment seems equipped with telocytes capable of stromal signaling. However, further studies are needed to assess whether the telocytes belong to a renewed cell population derived from circulating precursors.     

Temporal effects of cytokine treatment on lubricant synthesis and matrix metalloproteinase activity of fibroblast‐like synoviocytes

Fibroblast‐like synoviocytes (FLS) are major contributors to the composition and function of synovial fluid (SF). In disease, changes to important SF molecules such as hyaluronic acid (HA), lubricin, and numerous inflammatory markers contribute to a loss of SF functional properties. Previous studies characterized the ability of FLS to produce SF molecules in short‐term cultures using continuous cytokine supplementation. This study assessed the HA, lubricin, and matrix metalloproteinase‐2 (MMP‐2) secretion profile of FLS over 12 days of culture. FLS were subjected to continuous, intermittent, and sequential cytokine treatments of interleukin‐1 beta (IL‐1β), tumour necrosis factor‐alpha (TNF‐α), and transforming growth factor‐beta 1 (TGF‐β1). HA was assessed by an enzyme‐linked immunosorbent assay (ELISA) for content and agarose gel electrophoresis for molecular weight distribution. Relative lubricin content was determined by western blot. Pro MMP‐2 and active MMP‐2 were quantified by gelatin zymography. All intermittent and sequential treatments significantly increased secretion of high‐molecular‐weight (>3 MDa) HA for the duration of the culture. Sequentially treated groups elevated lubricin synthesis, whereas only groups receiving IL‐1β and TNF‐α for 2 days followed by TGF‐β1 for 1 day reduced active MMP‐2 to unstimulated control levels. These data provide important information on the long‐term functional potential of cytokine‐stimulated FLS and suggest that temporal regulation of cytokine exposure can be a powerful tool to guide healthy synovial secretions.     

TNF-α、IL-1β通过PKA通路诱导成纤维样滑膜细胞中力生长因子表达

目的 探讨炎症因子TNF-α、IL-1β、IL-6对力生长因子（mechano growth factor, MGF）表达的影响。方法 实验组细胞用浓度为25、50、100 ng/mL的TNF-α、IL-6或者是2.5、5.0、10 ng/mL的IL-1β处理12 h,抑制剂组在因子处理前用1.0 mmol KT5720预处理1 h,然后添加炎症因子刺激12 h,对照组细胞保持与实验组培养条件一致的情况下不添加因子刺激。因子刺激后,用定量PCR方法检测细胞中MGF表达量。结果25 ng/mL TNF-α和10 ng/mL IL-1β因子刺激滑膜成纤维细胞诱导MGF表达显著增加（P＜0.05）。IL-6对MGF表达无影响。1.0 mmol PKA通路抑制剂KT5720处理显著降低TNF-α和IL-1β诱导的MGF表达（P＜0.05）。结论 炎症因子TNF-α和IL-1β在浓度分别为25和10 ng/mL时能显著诱导滑膜成纤维细胞中MGF表达,其表达是通过PKA通路介导的。本研究对于促进MGF用于改善膝关节相关组织修复中的应力刺激不足有一定的现实意义。