TENDON CELLS--INTERACTIONS AND RESPONSES

描述：（改编自研究者摘要）屈肌腱 是用来将肌肉收缩的力量传递给骨骼， 影响肢体运动。 基质是主要的承重成分 然而，细胞是被动加载的。 肌腱表面 在滑动过程中受到剪应力，而整个肌腱 承受循环张力。 两个主要的细胞群体存在于屈肌 肌腱;表面腱外膜细胞（TSC）驻留在脉冲阻尼 半胶原蛋白和蛋白多糖和半脂质的环境， 肌腱内部的内部成纤维细胞（TIF）坐落在线性 阵列，这似乎是最佳的交界连接，在对齐 胶原纤维 申请人假设肌腱细胞可以接收并解释 机械信号通过与连接主管的相互通信 相邻的细胞。 细胞间的交流发生在目标 细胞释放细胞内钙储存，其信号被传播 通过IP 3依赖性机制通过间隙连接与相邻细胞连接。 用肝素处理靶细胞可防止信号传播， 阻断IP 3受体和氟烷治疗也阻断了 通过在缝隙连接处干扰来发出信号。 缝隙连接由以下组成 由6个相同的连接蛋白亚基组装的半通道连接蛋白。 鸟类细胞合成几种连接蛋白，其中连接蛋白43是 突出。 CXN-43磷酸化形式可能调节通道门控 打开/关闭状态。 调查人员发现， 肌腱细胞有连接蛋白43，它是磷酸化的内部 成纤维细胞，但不是表面滑膜细胞。 此外， 细胞在接种后可能需要时间（长达数天）来重建间隙 连接点的连接和信号后， 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，细胞迁移可能需要数天时间， 填充伤口可以重建它们的交流能力 申请人假设循环机械负荷将增加 肌腱中的间隙连接连接的数量，从而改善 细胞间信号传导 同样，固定化将 减少细胞间的通讯。 研究人员设计了一些实验来检验这些假设， 肌腱细胞在体内和体外模型的创伤和 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，并响应于机械刺激传播信号 一个细胞。 (2)在一个单独的或混合的 在新鲜分离的对数生长或静止细胞中培养TSC和TIF 施加+/-循环机械载荷，以刺激动态与静息 愈合的阶段，+/-运动;和（3）定量的量， 缝隙连接mRNA和连接蛋白43蛋白的合成率 静止期或对数期细胞+/-机械负荷和血清刺激， 并定量和关联连接蛋白43磷酸化状态， 交接能力 这些研究的结果应阐明如何 施加于肌腱或其孤立细胞的循环机械载荷影响 单个靶细胞响应单个机械刺激的能力 刺激并在愈合期间与邻近细胞相互交流。 一个重要的临床方面是， 被动渐进运动对损伤结缔组织的有益作用 可以鉴定恢复期的组织。

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.