【作者】 张艳君；

【导师】 宋国立；

【作者基本信息】 重庆大学 ， 生物医学工程， 2013， 博士

【摘要】 膝关节交叉韧带损伤后愈合能力差一直是骨科临床治疗面临的难题。因此从分子水平阐明交叉韧带难以愈合的原因至关重要。交叉韧带受到损伤的同时滑膜也会受到不同程度损伤。动物实验发现前交叉韧带(Anterior cruciate ligament, ACL)损伤后,与关节腔内其它组织(如：后交叉韧带(Posterior cruciate ligament, PCL)、半月板和软骨)相比,滑膜向关节液释放了最大量活化形式的基质金属蛋白酶(Matrix metalloproteinases, MMPs),因而被认为是交叉韧带损伤后关节腔内微环境的主要调节者。为了进一步更深入的探讨滑膜的关节腔微环境调控作用,我们体外模拟交叉韧带损伤后关节腔内微环境,在细胞水平上研究力-化学因素刺激下,滑膜成纤维细胞中赖氨酰氧化酶(Lysyl oxidases, LOXs)和MMPs的表达以及MMP-2活性。本文主要研究工作及结果如下：①TNF-α和IL-1β对滑膜成纤维细胞中LOXs和MMPs表达以及MMP-2活性的影响炎症因子被认为是炎症反应时期重要的化学调节者。研究结果显示lOng/ml TNF-α抑制了滑膜成纤维细胞中LOXs的表达10ng/ml IL-1β上调了LOX、LOXL-1和LOXL-3的表达,下调了LOXL-2和LOXL-4的表达。TNF-α和IL-1β分别上调了MMP-1,2,3的表达。TNF-α与IL-1β的结合不同程度地抑制了LOXs的表达,却以协同方式大大促进了MMP-1,2,3的表达。明胶酶谱实验指出TNF-α和IL-1β的结合进一步以剂量和时间依赖性形式诱导了MMP-2活性的增加,且MMP-2活性高于TNF-α和IL-1β的单独促进作用。炎症因子刺激下,滑膜成纤维细胞中表达失衡的LOXs和MMPs分泌进入关节液,造成关节腔中交叉韧带重塑过程的失衡,这可能是导致受损交叉韧带无法愈合的一个重要原因。说明受损交叉韧带无法愈合不仅与其自身因素有关,还与韧带损伤后关节腔内微环境的改变有关,其中滑膜对于关节腔内微环境的改变起着重要的调控作用。因此在研究交叉韧带修复工作中,滑膜是不能被忽视的。②损伤性力学拉伸作用下TNF-α对滑膜成纤维细胞中LOXs和MMPs表达以及MMP-2活性的影响在正常生理环境下,力学刺激是和关节腔内其它因子共同作用来引发损伤级联反应的。结果显示12%损伤性力学拉伸抑制了LOXs(除LOXL-2)的表达,却上调了MMP-1,2,3的表达。与10ng/ml的TNF-α相似,5ng/ml的TNF-α仍然抑制了LOXs的表达,促进了MMP-1,2,3的表达。在5ng/mlTNF-α存在的情况下,滑膜成纤维细胞受到损伤性力学拉伸的第1,2,3个小时里表现出的LOXs表达量的上调受到抑制,之后在6h时,LOXs表达量降到对照组水平以下。相反,损伤性力学拉伸与TNF-α以协同作用方式促进了MMP-1,2,3的表达以及MMP-2的活性。此外,体外划痕实验显示TNF-α抑制了细胞的迁移。在力学损伤和炎症因子共同作用下,滑膜成纤维细胞中表达失衡的LOXs和MMPs释放入滑液中引起关节腔微环境的变化,引起受损交叉韧带细胞外基质合成与降解的失衡,不利于韧带修复。以上结果说明除了交叉韧带自身因素外,关节腔中微环境的改变也影响着韧带的愈合能力。此外,研究结果还指出滑膜成纤维细胞对力学因子和炎症因子非常敏感,并且在调节关节腔微环境中起着重要作用。因此在研究交叉韧带修复工作中,滑膜是不能被忽视的。③损伤性力学拉伸和TGF-β1对滑膜成纤维细胞中LOXs和MMP-1,2,3表达以及MMP-2活性的影响在韧带愈合的炎症反应期,不仅炎症因子(TNF-α和IL-1β)而且生长因子(如TGF-β1)也参与了韧带的修复。结果显示损伤性力学拉伸抑制了LOXs(除LOXL-2)的表达,而TGF-β1上调了LOXs的表达。损伤性力学拉伸和’TGF-β1分别上调了MMP-1,2,3的表达。损伤性力学拉伸与TGF-β1共同作用,促进了LOXs(除LOXL-1)和MMP-1,2,3的表达,并且MMPs的上调程度大于LOXs的上调程度。此外,损伤性力学拉伸与TGF-β1结合,以时间依赖性方式大大促进了MMP-2的活性,明显超过了力因子单独对MMP-2的作用。在力学损伤和生长因子共同作用下,相对于LOXs的表达水平,滑膜成纤维细胞中更高的MMPs表达和活性破坏了交叉韧带细胞外基质重塑过程的平衡状态,不利于韧带修复。以上结果除了说明交叉韧带受损后关节腔微环境改变对韧带愈合的不利影响外,也说明滑膜在关节腔微环境调控过程中起着重要作用,并且其作用的发挥受力因子和生长因子的共同影响。因此,滑膜在韧带愈合过程中的作用越来越重要,不能被忽视。④NF-κB信号通路介导了损伤性力学拉伸和TGF-β1对滑膜成纤维细胞中MMP-2活性的调节过程利用信号通路抑制剂来研究损伤性力学拉伸和TGF-β1调控滑膜成纤维细胞中MMP-2表达的主要信号通路。结果显示由于NF-κB信号通路抑制剂Bay11-7082和Bay11-7085的加入,损伤性力学拉伸和TGF-β1诱导的滑膜成纤维细胞中MMP-2的活性被抑制。⑤为了更真实的模拟体内环境,实验建立共培养体系,研究共培养环境下,滑膜细胞对PCL细胞中LOXs的直接性的调节作用,结果显示相比单培养而言,共培养促进了PCL细胞中LOXs的表达。这一结果直接表明了滑膜与PCL之间有交流,并且更加说明滑膜作为微环境调节者参与了交叉韧带的愈合。综上所述,交叉韧带损伤后关节腔微环境的改变可能是导致其自身修复失败的重要原因,并且滑膜通过对关节腔内微环境的调控参与了交叉韧带的愈合过程。因此通过调节滑膜中LOXs和MMPs的表达来改善关节腔微环境对于提高交叉韧带愈合能力具有重要意义。更多还原

【Abstract】 Injured knee joint cruciate ligament does not self-heal satisfactorily making it as one of the most challenging and intractable clinical problem today. This raises the necessity to detail the reason why the injured cruciate ligament hardly repair.The damage of cruciate ligament is always accompanied by the injury of synovium. Previous ex vivo studies showed that the synovium has the greatest ability for converting the72kDa pro-MMP-2to the62kDa active-MMP-2and releasing MMP-2into the synovial fluids compared with other intraarticular tissues (such as posterior cruciate ligament (PCL), meniscus and cartilage). Therefore the synovium is considered to be the major regulator of the microenvironment in the joint cavity after cruciate ligament injury. In order to further explore the role of synovium in regulating the microenvironment of joint cavity after cruciate ligament injury, we mimicked the microenvironment of joint cavity after cruciate ligament injury and study the effects of mechanical-chemical factors on expressions of LOXs (Lysyl oxidases, LOXs) and MMPs and MMP-2activity in synovial fibroblasts at the cellular level. The main research and results are as follows:①Combined effects of TNF-a and IL-1β on LOXs and MMPs expressions in synovial fibroblastsProinflammatory factors are considered important chemical mediators in the acute inflammatory phase of wound healing. The results showed that lOng/ml TNF-α alone down-regulated the expressions of LOXs. lOng/ml IL-1β up-regulated the expressions of LOX、LOXL-1and LOXL-3, down-regulated the expressions of LOXL-2and LOXL-4. Both TNF-a and IL-1β increased the expressions of MMP-1,2,3. When the two inflammatory factors were mixed, expressions of LOXs were inhibited differently. However, the combination of TNF-a plus IL-1β caused a greater increase in the expressions of MMP-1,-2, and-3relative to TNF-a and IL-1β alone. Zymography assays showed that the combination of TNF-a and IL-1β increased MMP-2activity in a dose and time dependent manner and the MMP-2activity was much higher than MMP-2activity induced by TNF-α and IL-1β alone. In the presence of proinflammatory factors, the LOXs and MMPs showing a state of imbalanced expressions in synovial fibroblasts were released into synovial fluids and alter the balance of cruciate ligament healing, which may be one of the important reasons why cruciate ligament cannot be repaired. These results suggest that besides self-factors of cruciate ligament, microenvironment in knee joint cavity also affect the cruciate ligament’s healing ability and synovium plays an important role in regulating the microenvironment in the joint cavity and cannot be ignored in the healing process of cruciate ligament.②Effects of TNF-a on expressions of LOXs and MMPs in synovial fibroblasts under injurious mechanical stretchIn the real physiological situation, the stretching force may exert its effect together with other factors to induce the injurious cascade. The results showed that12%injurious stretch down-regulated the expressions of LOXs (except LOXL-2) and up-regulated the expressions of MMP-1,2,3. As above lOng/ml TNF-α,5ng/ml TNF-α also down-regulated the expressions of LOXs and up-regulated the expressions of MMP-1,2,3. In the presence of TNF-a, the12%injurious stretch-induced up-regulation of LOXs at1,2and3h was significantly inhibited and decreased further below control after6h. In contrast, injurious stretch had a synergistic effect with TNF-a on increasing expressions of MMP-1,2,3and MMP-2activity. In addition, in vitro wound healing assay revealed that TNF-a inhibited the migration of synovial fibroblasts. Under the stimulation of injurious stretch and TNF-a, the LOXs and MMPs of imbalance in synovial fibroblasts released into synovial fluids and caused the change of microenvironment in knee joint cavity which would promoted the ECM degradation of cruciate ligament and inhibit the ligament repair. These results suggest that besides self-factors of cruciate ligament, microenvironment in knee joint cavity also affect the cruciate ligament’s healing ability. In addition, these results further indicate that synovial fibroblasts are very sensitive to mechanical injury and proinflammatory factors and plays an important role in regulating the microenvironment in the joint cavity and cannot be ignored in the healing process of cruciate ligament.③Combined effects of mechanical stretch and TGF-β1on LOXs and MMPs expressions in synovial fibroblastsIn the inflammatory phase of ligament healing, not only proinflammatory factors (such as TNF-a and IL-1β) but also growth factors (such as TGF-β1) participate in the ligament repair. The results showed that injurious stretch down-regulated the expressions of LOXs (except LOXL-2), however, TGF-β1up-regulated the expressions of LOXs. Both injurious stretch and TGF-β1alone increased the expressions of MMP-1, 2,3. When injurious stretch and TGF-β1were mixed, expressions of LOXs and MMP-1,2,3were up-regulated and the elevated level of MMP-1,2,3was higher than LOXs. In addition, the combination of injurious stretch and TGF-β1increased MMP-2activity in a time dependent manner and the MMP-2activity was much higher than MMP-2activity induced by injurious stretch alone. Under the stimulation of injurious stretch and TGF-β1the much higher expressions of MMPs in synovial fibroblasts compared to LOXs promoted the ECM degradation of cruciate ligament, which would inhibit the ligament repair. These results suggest that change of microenvironment in knee joint cavity after cruciate ligament injury affects the cruciate ligament’s healing ability. The synovial fibroblasts are very sensitive to mechanical injury and growth factors, and further indicate that synovium plays an important role in regulating the microenvironment in the joint cavity after cruciate ligament injury must be considered seriously.④NF-κB pathway is involved in the injurious stretch and TGF-β1induction of MMP-2in human synovial fibroblastsThe signal pathways underlining the injurious stretch and TGF-β1-induced MMP-2activity in synovial fibroblasts were investigated through signal pathway inhibitor. These results suggest that the induced MMP-2by injurious stretch and TGF-β1was inhibited significantly due to the addition of NF-κB pathway inhibitor Bay11-7082and Bay11-7085.⑤In order to mimicked the microenvironment of joint cavity more really, we established coculture system and investigated the direct regulation of synovial fibroblasts on PCL fibroblasts. The results showed that coculture promoted the expressions of LOXs in PCL fibroblasts compared mono-culture.These results suggest that synovial fibroblasts and PCL fibroblasts do exist interaction and crosstalk, and further indicate that synovium participates in the healing process of cruciate ligament as microenvironment regulator in the joint cavity.In summary, these results suggest that the change of microenvironment in knee joint cavity after cruciate ligament injury may be one of the important reasons why cruciate ligament cannot be repaired and synovial fibroblasts participate in the healing process of cruciate ligament as microenvironment regulator. Therefore, improving the microenvironment in the joint cavity after cruciate ligament injury through regulating LOXs and MMPs expressions in synovial fibroblasts would has important significance for cruciate ligament repair.更多还原