TENDON CELLS--INTERACTIONS AND RESPONSES

肌腱细胞——相互作用和反应

• 批准号: 2413968
• 负责人: ALBERT J BANES
• 金额: $ 17.78万
• 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 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2413968
• 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：（改编自研究者摘要）屈肌腱 旨在将肌肉收缩的力量传递到骨骼 影响肢体运动。 基体是主要的承重构件 肌腱；然而，电池是被动加载的。 肌腱表面 滑行过程中受到剪应力，而整个肌腱 受到循环张力。 屈肌中存在两种主要细胞群 肌腱;表面表膜细胞 (TSC) 驻留在脉冲抑制中 一半胶原蛋白、蛋白多糖和一半脂质的环境，以及 肌腱内部的内部成纤维细胞（TIF）呈线性分布 阵列，对于连接连接来说是最佳的，在对齐的中间 胶原纤维。 申请人假设肌腱细胞可以接收和解释 通过与连接主管相互通信的机械信号 相邻的细胞。 细胞间通讯发生在目标之后 细胞释放细胞内钙储备，其信号被传播 通过 IP3 依赖性机制通过间隙连接到达邻近细胞。 用肝素处理靶细胞可防止信号传播 阻断 IP3 受体和氟烷治疗也会阻断 通过干扰间隙连接处发出信号。 间隙连接由以下部分组成 半通道连接子由 6 个相同的连接蛋白亚基组装而成。 鸟类细胞合成多种连接蛋白，其中连接蛋白43是 著名的。 CXN-43 磷酸化形式可能调节通道门控 到打开/关闭状态。 调查人员发现，鸟类 肌腱细胞有连接蛋白 43，它在内部被磷酸化 成纤维细胞，但不是表面滑膜细胞。 此外，培养肌腱 细胞在电镀后可能需要时间（最多几天）来重新建立间隙 连接点连接以及在连接后相互发送信号的能力 机械刺激。 因此，重建间隙连接能力 可能需要新的合成和磷酸化状态的改变。 在愈合的肌腱中，细胞迁移可能需要几天的时间 填充伤口可以重建他们的相互交流能力。 申请人假设循环机械负载会增加 肌腱中间隙连接连接的数量导致改善 细胞间信号随时间变化。 同样，固定化也会 减少细胞间通讯。 研究人员设计了实验来检验这些假设 损伤和体外模型中的肌腱细胞 机械扰动具有以下具体目的：（1）测试 正常和受伤肌腱中的细胞释放 [Ca2+}i 并传播响应机械刺激的信号 到单个细胞。 (2) 单独或混合测试相同的响应 在新鲜分离的对数生长或静止细胞中培养 TSC 和 TIF +/- 施加循环机械负载，以刺激动态与静态 愈合阶段，+/- 运动； (3) 定量数量和 间隙连接 mRNA 和连接蛋白 43 蛋白的合成率 静止或对数期细胞+/-机械负荷和血清刺激， 并对连接蛋白 43 磷酸化状态进行定量和关联 连接能力。 这些研究的结果应该阐明如何 施加于肌腱或其孤立细胞的循环机械负荷会影响 单个靶细胞对单个机械反应的能力 在愈合过程中刺激并与邻近细胞相互交流。 一个重要的临床方面是，其背后的机制 被动渐进运动对受伤结缔组织的有益影响 可以识别恢复期间的组织。