TENDON CELLS--INTERACTIONS AND RESPONSES

描述：(改编自《调查者摘要》)屈肌腱 旨在将肌肉收缩的力量传递到骨骼 影响肢体运动。母体是主要的承重部件 肌腱；然而，细胞是被动加载的。肌腱表面 在滑行过程中受到剪应力，而整个肌腱 接收循环张力。屈肌中存在两个主要的细胞群 肌腱；驻留在脉搏中的表面外膜细胞(TSC) 一半的胶原蛋白、蛋白多糖和一半的脂肪，以及 肌腱内部成纤维细胞呈线状排列 阵列，看起来是最适合连接的阵列，在排列的 胶原纤维。 申请者假设肌腱细胞可以接收和解释 通过与连接能力相互通信的机械信号 相邻的细胞。细胞间通信发生在目标之后 细胞释放细胞内的钙储存，其信号被传播 通过缝隙连接通过依赖于IP3的机制传递给相邻细胞。 用肝素处理靶细胞可通过以下途径阻止信号传播 阻断IP3受体和氟烷治疗也可阻断 通过在缝隙连接处干扰来发出信号。缝隙连接由以下部分组成 半通道连接蛋白由6个相同的连接蛋白亚基组装而成。 禽类细胞合成多种连接蛋白，其中连接蛋白43是 很突出。CXN-43磷酸化形式可能调节通道门控 设置为打开/关闭状态。调查人员发现，禽类 肌腱细胞有连接蛋白43，它在体内被磷酸化。 成纤维细胞，而不是表面滑膜细胞。此外，培养的肌腱 细胞在培养后可能需要一段时间(最多几天)才能重新建立缺口 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，重新建立缝隙连接能力 可能需要在磷酸化状态下进行新的合成和改变。 在愈合的肌腱中，可能需要几天时间才能迁移细胞 填充伤口可以重新建立他们相互交流的能力。 申请人假设循环机械载荷将增加 肌腱中缝隙连接的数量得到改进 随时间变化的细胞间信号传递。同样，固定化将 减少细胞间的通讯。 研究人员设计了实验来验证这些假设 肌腱细胞在体内和体外损伤模型中的作用 具有以下特定目的的机械扰动：(1)测试 正常肌腱和损伤肌腱中细胞的能力 [Ca~(2+)]i和传播信号以响应机械刺激 只有一个细胞。(2)在单独或混合的情况下测试相同的响应 原木生长细胞和静止细胞中TSC和TIF的培养 施加+/-循环机械载荷，以刺激动态与静息 愈合阶段，+/-运动；以及(3)量化数量和 细胞间隙连接蛋白43蛋白和缝隙连接蛋白的合成速率 静止期或对数期细胞+/-机械负荷和血清刺激， 并量化和关联连接蛋白43的磷酸化状态和 结合部能力。这些研究的结果应该阐明 施加于肌腱或其孤立细胞的循环机械载荷影响 单个目标细胞对单个机械信号作出反应的能力 在愈合过程中与邻近细胞进行刺激和相互交流。 一个重要的临床方面是，潜在的机制 被动递进运动对损伤的结缔组织的有益作用 恢复期的组织可以被识别出来。

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.