TENDON CELLS--INTERACTIONS AND RESPONSES

肌腱细胞——相互作用和反应

• 批准号: 2079262
• 负责人: ALBERT J BANES
• 金额: $ 17.37万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2079262
• 关键词: Aves; calcium flux; collagen; fibroblasts; gap junctions; growth factor; lipids; mechanical stress; membrane channels; mixed tissue /cell culture; phosphorylation; protein biosynthesis; proteoglycan; tendon injury; wound healing

DESCRIPTION: (Adapted from the Investigator's Abstract) Flexor tendons are designed to transmit the force of muscle contraction to bone to effect limb movement. The matrix is the major load-bearing component of tendon; however the cells are passively loaded. The tendon surface is subjected to shear stress during gliding, while the whole tendon receives cyclic tension. Two major cell populations exist in flexor tendon; the surface epitenon cells (TSC) residing in a pulse dampening milieu of half collagen and proteoglycan and half lipid, and the internal fibroblasts (TIF) of the tendon interior nestled in linear arrays, that appear optimal for junctional connectivity, amidst aligned collagen fibers. The applicants hypothesize that tendon cells can receive and interpret mechanical signals by intercommunicating with junctionally competent neighboring cells. Intercellular communication occurs after a target cell releases intracellular calcium stores whose signal is propagated to neighboring cells through gap junctions by an IP3-dependent mechanism. Treatment of target cells with heparin prevents signal propagation by blocking IP3 receptors and treatment with halothane also blocks the signal by interfering at gap junctions. Gap junctions are comprised of hemichannel connexons assembled from 6 identical connexin subunits. Avian cells synthesize several connexins, of which connexin 43 is prominent. The CXN-43 phosphorylation forms may regulate channel gating to the open/closed states. The investigators have found that avian tendon cells have connexin 43 and that it is phosphorylated in internal fibroblasts, but not surface synovial cells. Moreover, cultured tendon cells can require time (up to days) after plating to reestablish gap junction connections and the ability to signal each other after a mechanical stimulus. Therefore, reestablishing gap junction competency may require new synthesis and alteration in the phosphorylation state. In a healing tendon, days may be required before migrating cells populating a wound can reestablish their ability to intercommunicate. The applicants hypothesize that cyclic mechanical load will increase the number of gap junction connections in tendon resulting in improved intercellular signalling with time. Likewise, immobilization will decrease intercellular communication. The investigator have designed experiments to test these hypotheses in tendon cells in both in vivo and in vitro models of wounding and mechanical perturbation with the following specific aims: (1) to test the ability of cells in normal and wounded tendon to mount a release of [Ca2+}i and propagate the signal in response to a mechanical stimulus to a single cell. (2) to test the same response in a separate or mixed culture of TSC and TIF in freshly isolated log growth or quiescent cells +/- cyclic mechanical load applied, to stimulate dynamic vs resting phases of healing, +/- motion; and (3) to quantitate the amount and synthetic rates of gap junction mRNA and connexin 43 protein in quiescent or log phase cells +/- mechanical load and serum stimulation, and quantitate and correlate the connexin 43 phosphorylation state and junctional competency. Results of these studies should elucidate how cyclic mechanical loading applied to tendon or its isolated cells affects the ability of a single target cell to respond to a single mechanical stimulation and intercommunicate with neighboring cells during healing. An important clinical aspect is that the mechanism underlying the beneficial effects of passive progressive motion to injured connective tissues during convalescence may be identified.

描述：（根据调查员的摘要改编）屈肌肌腱 旨在将肌肉收缩的力传递给骨骼 影响肢体运动。 矩阵是主要承载组件 肌腱；但是，细胞被动加载。 肌腱表面 在滑行过程中受到剪切应力，而整个肌腱 接收环状张力。 屈肌中存在两个主要的细胞种群 肌腱;居住在脉冲抑制的表面上皮细胞（TSC） 一半胶原蛋白和蛋白聚糖和一半脂质的环境，以及 内部肌腱内部的内部成纤维细胞（TIF） 阵列，对于连通连通性似乎是最佳的，在对齐中 胶原纤维。 申请人假设肌腱细胞可以接收和解释 通过与连接胜任的互通来机械信号 相邻的细胞。 细胞间通信发生在目标之后 细胞释放信号传播的细胞内钙存储 通过依赖IP3的机制通过间隙连接到相邻的细胞。 用肝素处理靶细胞可防止信号传播 阻断IP3受体和卤烷处理也可以阻止 通过干扰间隙连接的信号。 差距连接由 半通道连接子来自6个相同的连接蛋白亚基。 鸟类细胞合成几种连接素，其中连接蛋白43为 著名的。 CXN-43磷酸化形式可能调节通道门控 到开放/关闭状态。 调查人员发现鸟类 肌腱细胞具有连接蛋白43，并在内部磷酸化 成纤维细胞，但没有表面滑膜。 而且，培养的肌腱 电镀后需要时间（最多几天）才能重新建立差距 连接连接和在 机械刺激。 因此，重新建立间隙连接能力 可能需要在磷酸化状态下进行新的合成和改变。 在愈合肌腱中，可能需要天数 填充伤口可以重新建立他们的间交流能力。 申请人假设环状机械负载将增加 肌腱中的间隙连接数量，导致改善 随时间的细胞间信号传导。 同样，固定化也会 减少细胞间通信。 研究人员设计了实验来检验这些假设 体内和体外受伤的体外模型中的肌腱细胞和 机械扰动具有以下特定目的：（1）测试 细胞在正常和受伤肌腱中的能力安装 [Ca2+} i并响应机械刺激而传播信号 到一个单元格。 （2）在单独或混合中测试相同的响应 新鲜分离的对数生长或静态细胞中TSC和TIF的培养 +/-施加了循环机械载荷，以刺激动态与静止 治愈的阶段，+/-运动； （3）定量金额和 间隙连接mRNA和连接蛋白43蛋白的合成速率 静态或对数相细胞+/-机械负荷和血清刺激， 并定量并关联连接蛋白43磷酸化状态和 连接能力。 这些研究的结果应阐明如何 适用于肌腱或其孤立细胞的环状机械负荷会影响 单个目标细胞对单个机械做出反应的能力 在愈合过程中，刺激并与邻近细胞进行了互通。 一个重要的临床方面是， 被动渐进运动对受伤结缔组织的有益影响 可以鉴定在康复期间的组织。