54 The Stem Cell Niche In Tendon And Ligament - Investigating Alterations With Ageing And Disease
54 The Stem Cell Niche In Tendon And Ligament - Investigating Alterations With Ageing And Disease
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54 肌腱和韧带中的干细胞生态位 - 研究衰老和疾病引起的变化
DOI：
10.1136/bjsports-2014-094114.54
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发表时间：
2014
影响因子：
18.4
通讯作者：
Lee K
中科院分区：
文献类型：
--
作者：
Lee K
韧带和肌腱容易因老化和损伤而退化，目前的治疗方法大多无效。肌腱内具有干细胞样特征的细胞群的鉴定（Bi，2007）具有肌腱和韧带再生的潜力。肌腱干细胞分化为肌腱细胞（Zhang，2010）;肌腱内的主要细胞类型，负责产生细胞外基质（ECM）。局部干细胞环境（小生境）对于许多组织中的干细胞维持和功能至关重要，并且特别是已显示腱生蛋白C在干细胞小生境中发挥重要作用（Garcion，2004）。肌腱和韧带由束和束间基质（IFM）组成，其组成差异很大，在组织内提供确定的小生境。本研究的目的是观察马肌腱和犬韧带中干细胞龛的ECM成分，这些干细胞龛容易发生与年龄相关的退化。本研究的目标是产生anin vitroenvironment干细胞模仿干细胞龛，用于治疗肌腱和韧带diseases. MethodsPuzzious干细胞分离从马浅趾屈肌腱（SDFT）和犬前交叉韧带（ACL）通过低密度平板和差分粘附塑料和纤维连接蛋白基板。使用间充质干细胞标志物CD 90、CD 73和CD 105的抗体通过流式细胞术以及干细胞和肌腱生成标志物的qRT-PCR分析细胞。使用放射性同位素标记分析成纤维细胞和干细胞龛的ECM组分。在提取ECM之前，用14 C标记的氨基酸标记细胞以特异性标记新合成的胶原性（脯氨酸）和非胶原性（赖氨酸/精氨酸）ECM。免疫组化和组织学进行分析SDFT.ResultsTendon和韧带细胞形成的低密度平板后的结构和组成，但只有韧带细胞形成的差异粘附到纤维连接蛋白后的集落。肌腱细胞亚群表达CD 90在新鲜分离的细胞和假定的干细胞，但CD 105和CD 73阴性。推定肌腱干细胞，分离的差异纤连蛋白粘附没有表现出增加表达的干细胞标记物相比，肌腱细胞。然而，与韧带细胞相比，在假定的韧带干细胞中干细胞标记物的表达显著增加。肌腱细胞和假定的肌腱干细胞（分离的低密度平板）标记与14 C-标记的氨基酸都显示出类似的标记配置文件。SDFT组织的组织学分析突出了肌腱的不同结构和组成，腱生蛋白C的表达局限于IFM（见图1）。已发表的人类和其他哺乳动物肌腱干细胞分离技术似乎对马肌腱干细胞的分离无效。或者，马肌腱细胞群可能由处于不同分化阶段的细胞的异质混合物组成。我们目前正在优化干细胞分离技术，并进行三系分化试验。其他物种的肌腱干细胞表现出三系分化，但马肌腱干细胞可能仅限于肌腱分化。未来的实验旨在通过质谱和肌腱和软骨的微阵列比较来识别干细胞龛的ECM成分。
IntroductionLigament and tendon are prone to degeneration through ageing and injury and current therapies are largely ineffective. The identification of a cell population within tendon with stem cell-like characteristics (Bi, 2007) holds potential for regeneration of tendon and ligament. Tendon stem cells differentiate into tenocytes (Zhang, 2010); the predominant cell type within tendon, responsible for producing extracellular matrix (ECM). The local stem cell environment (niche) is vital for stem cell maintenance and function in many tissues, and tenascin C in particular has been shown to play an important role within stem cell niches (Garcion, 2004). Tendon and ligament are composed of fascicles and interfascicular matrix (IFM) which vary considerably in composition providing definitive niches within the tissue. This study aims to characterise ECM components of the stem cell niche in equine tendon and canine ligament, which are prone to age-related degeneration. The goal of this research is to produce anin vitroenvironment for stem cells which mimics the stem cell niche, for treatment of tendon and ligament disease.MethodsPutative stem cells were isolated from equine superficial digital flexor tendon (SDFT) and canine anterior cruciate ligament (ACL) by low-density plating and differential adhesion to plastic and fibronectin substrates. Cells were analysed by flow cytometry using antibodies to mesenchymal stem cell markers CD90, CD73 and CD105, as well as qRT-PCR for stem cell and tenogenic markers. ECM components of the fibroblast and stem cell niche were analysed using radioisotope labelling. Cells were labelled with14C-labelled amino acids to specifically label newly synthesised collagenous (proline) and non-collagenous (lysine/arginine) ECM, prior to extraction of ECM. Immuno-histochemistry and histology were conducted to analyse the structure and composition of SDFT.ResultsTendon and ligament cells formed colonies after low-density plating, however only ligament cells formed colonies after differential adhesion to fibronectin. A subpopulation of tendon cells expressed CD90 in both freshly isolated cells and putative stem cells, but were CD105 and CD73 negative. Putative tendon stem cells, isolated by differential fibronectin adhesion did not exhibit increased expression of stem cell markers when compared with tenocytes. However there was a significant increase in expression of stem cell markers in putative ligament stem cells compared with ligamentocytes. Tenocytes and putative tendon stem cells (isolated by low-density plating) labelled with14C-labelled amino acids both displayed similar labelling profiles. Histological analysis of SDFT tissue highlighted the varied structure and composition of tendon, with tenascin C expression confined to IFM (see Figure.1).ConclusionThe absence of stem cell marker expression in putative stem cell populations indicates that further testing of stem cell isolation procedures is required. Published techniques for tendon stem cell isolation in humans and other mammals do not appear to be effective for isolation of equine tendon stem cells. Alternatively it is possible that the equine tendon cell population consists of a heterogenous mixture of cells at different stages of differentiation. We are currently optimising stem cell isolation techniques and conducting tri-lineage differentiation assays. Tendon stem cells in other species show tri-lineage differentiation, however it is possible that equine tendon stem cells may be restricted to tenogenic differentiation. Future experiments aim to identify ECM components of the stem cell niche by mass spectrometry and microarray comparison of tendon and …