【作者】 王业全；

【导师】 杨力；

【作者基本信息】 重庆大学 ， 生物医学工程， 2011， 博士

【摘要】 膝关节前交叉韧带(Anterior cruciate ligament,ACL)损伤是一种较常见的运动性损伤,韧带的撕裂或断裂都会造成膝关节不稳,长期损伤会导致膝关节退行性病变,甚至骨关节炎等并发症。当前临床治疗面临最大的挑战就是ACL修复能力较弱,损伤后不能像内侧副韧带一样能够自身功能性修复。除了ACL组织自身存在的先天遗传性差异外,其损伤后关节腔微环境的变化也可能是调节损伤的ACL修复的关键因素。因此本文着重从ACL损伤后的关节腔微环境入手,探讨导致其自身修复失败的主要原因。本文的主要工作及结果如下:1)动物实验探讨ACL损伤后关节腔微环境的变化利用大鼠ACL瞬时扭转损伤模型发现在ACL损伤后关节液中炎症因子IL-1β、IL-6和TNF-α的表达都显著升高。MMP-2以及整体MMPs家族的活性都随时间依赖性增加。体外组织培养实验发现关节腔内组织都向关节液中释放大量的MMP-2,其中以滑膜组织的作用最为显著。因此我们认为在ACL损伤后也会伴随伤口愈合机制的发生,关节腔内组织尤其滑膜组织以及炎症因子所导致的关节液中大量MMPs的累积可能是ACL损伤后不能修复的一个重要的原因所在。2)体外实验探讨关节腔微环境因素对MMP-2表达的影响①机械应力与炎症因子对滑膜成纤维细胞MMP-2活性的影响动物实验发现在ACL损伤后滑膜组织可能是关节液中MMPs的主要调节者,体外实验进一步阐明机械应力与炎症因子是否会诱导滑膜成纤维细胞MMP-2的活性表达,以及力-化学因素在调节MMP-2表达中所起的不同作用。实验结果表明机械应力强度的增加能够显著增强MMP-2的活性表达,炎症因子TNF-α以时间以及剂量依赖形式诱导MMP-2活性的增加,然而IL-1α的作用则不显著。当机械应力与炎症因子共同作用于滑膜成纤维细胞时,则对MMP-2活性的增加有着显著的协同作用。整体MMPs活性检测表明机械应力能够诱导MMPs活性的增加而炎症因子则不能,这也意味着机械应力诱导的MMPs是以酶原形式MMPs为主,而炎症因子诱导的则主要是活化形式MMPs。滑膜组织对机械损伤以及炎症因子非常敏感,并且在调节关节液中MMPs的表达起着至关重要的作用,因此在研究膝关节损伤与修复的过程中滑膜组织是不容忽视的。②机械应力作用下TNF-α与IL-1β对ACL成纤维细胞MMP-2表达的影响ACL损伤后关节腔内伴随着炎症因子、机械应力以及一些修复因子的复杂的动态变化。损伤的关节腔内组织导致各种炎症因子、细胞因子以及基质金属蛋白酶的累积。因此膝关节损伤后关节液中大量MMPs的累积被认为是ACL修复能力弱的关键因素,然而这些因素对ACL成纤维细胞MMP-2活性影响的具体作用还不明了。因此,为了探讨机械损伤应力与炎症因子对MMP-2表达的共同作用,我们进行体外模拟ACL损伤后的关节腔微环境,来阐明力-化学因素对ACL成纤维细胞MMP-2活性表达的影响。结果表明TNF-α与IL-1β能够以时间以及剂量依赖的形式增加MMP-2的活性,其中以IL-1β的作用最为显著。机械应力与炎症因子共同作用能够进一步增强MMP-2的活性。实验结果阐明机械损伤与炎症因子的共同作用对ACL成纤维细胞MMP-2的活性表达起着重要的调控作用。③机械应力与低氧对HIF-1α和MMP-2表达的影响除了炎症反应,在ACL损伤后低氧也是调控关节腔微环境的一个重要因素,然而低氧在ACL损伤后作用的机理还不清楚。因此我们在体外模拟低氧以及机械损伤微环境来探讨ACL成纤维细胞在机械应力损伤后低氧对MMP-2、VEGF、CTGF以及HIF-1α表达的影响。实验结果发现机械应力损伤与低氧分别作用于ACL成纤维细胞时能够增加MMP-2、VEGF、CTGF以及HIF-1α表达,但是当两者共同作用时则能进一步增加MMP-2的表达,而下调VEGF、CTGF以及HIF-1α表达。这一结果说明高表达的MMP-2以及低表达的VEGF、CTGF以及HIF-1α对ACL成纤维细胞不利,影响ACL修复能力。而且ACL损伤导致的低氧微环境也可能是组织退行性病变的一个诱因所在。3) NF-κB信号通路介导ACL成纤维细胞MMP-2的表达利用信号通路抑制剂来探讨ACL成纤维细胞损伤后调控MMP-2的表达的主要信号通路。实验结果发现NF-κB信号通路抑制剂(Bay11-70082)能够显著抑制MMP-2的表达,而PD98059 (ERK信号通路抑制剂),SP600125 (JNK信号通路抑制剂)以及SB203850 (p38信号通路抑制剂)则对MMP-2的活性没有显著影响。综合以上结论我们推测,ACL损伤后关节腔微环境的变化可能是导致其自身修复失败的主要原因,通过抑制MMPs的活性以及改善关节腔微环境有利于损伤的ACL修复。更多还原

【Abstract】 Injury to the anterior cruciate ligament (ACL) causes knee instability, pain, and may result in progressive degeneration and premature osteoarthritis. An injured ACL does not heal satisfactorily as compared to other joint tissues such as medial collateral ligament (MCL), making it as one of the most challenging and intractable clinical problem today. Apart from these intrinsic differences, microenvironment in knee joint cavity also regulates the ligament’s healing ability. Therefore, in this study, we focus on microenvironment of joint cavity to explore the cause of ACL repair failure. The main experiments and results are as follows:1) Contributions of different intraarticular tissues to the acute phase elevation of synovial fluid MMP-2 following rat ACL ruptureWith a rat ACL rotating injury model, we found that levels of IL-1β, IL-6, and TNF-αwere significantly higher in synovial fluids after ACL injury. MMP-2 activity and global MMP activity in synovial fluids also increased significantly in a time-dependent manner. Ex vivo studies showed that all tissues contributed to the elevation of MMP-2 in synovial fluids especially synovium and the injuried ACL. We concluded that although the regular wound healing mechanism also accurs after ACL injury, accumulation of MMP activity in the synovial fluids, due to all of the intraarticular tissues, may be at least one of the important reasons why an injured ACL cannot be repaired.2) The effects of microenvironment factors on MMP-2 production①Differential MMP-2 activity induce by mechanical compression and inflammatory factors in synoviocytesSynovium may be the major regulator of MMPs in joint cavity after injury, in vitro study to determine whether mechanical injury and inflammatory factors will induce MMP-2 production in synoviocytes. We found mechanical compression increased the MMP-2 production. In addition, TNF-αcan also elevate the activity of MMP-2 in a dose dependent manner, while IL-1αdoes not. However, mixture of these two factors dramatically increased MMP-2 production. In addition, mechanical injury had a strong synergistic effect on MMP-2 production with IL-1α, TNF-αand their mixture. The generic MMP activity assay revealed that mechanical compression increased the generic activity by only , APMA treatment increased the generic activity of MMPs induced by compression but not inflammatory factors, which indicated that compression would induced MMPs in pro-form while inflammatory factors induced MMPs mostly in active-form.②Combined effects of TNF-αand IL-1βon MMP-2 production in ACL fibroblasts under mechanical stretchThe dynamics between inflammatory factors, mechanical stress and healing factors, in an intra-articular joint, are very complex after injury. Injury to intra-articular tissue (anterior cruciate ligament, ACL, synovium) results in accumulation of various pro-inflammatory factors, cytokines and metallo-proteases. Although the presence of increased amounts of MMPs in the joint fluid after knee injury is considered the key factor for ACL poor healing ability; however, the exact role of collective participants of the joint fluid on MMP-2 activity and production has not been fully studied yet. To investigate the combined effects of mechanical injury and inflammation on induction of MMP-2; we mimicked the microenvironment of joint cavity after ACL injury. The results show that TNF-αand IL-1βelevate the activity of MMP-2 in a dose and time dependent manner. In addition, mechanical stretch further enhances the MMP-2 protein levels with TNF-α, IL-1β, and their mixture. Our results conclude that mechanical injury and inflammatory factors collectively induce increased MMP-2 production in ACL fibroblasts.③Mechanical stretch and HIF-1αaffects vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) in ACL fibroblastsHypoxia plays important role in regulating microenvironment of joint cavity after ACL injury, however, its role in mechanical injury is yet to be examined fully in ACL fibroblasts. In this study, we investigated the influence of hypoxia on matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and hypoxia inducible factor-1 alpha (HIF-1α) expression in ACL fibroblasts after mechanical injury. The real-time PCR results show that mechanical stretch or hypoxia increases the expression of MMP-2, VEGF, CTGF and HIF-1α; however, the synergistic effects of mechanical stretch and hypoxia increased MMP-2 production but decreased the VEGF, CTGF and HIF-1αexpression. Western blot analysis and ELISA also confirmed these results. Our results demonstrate that increased levels of MMP-2 and decreased levels of HIF-1α, VEGF and CTGF are detrimental to ACL fibroblasts hence affecting their healing ability and may be one of the prime reason’s of tissue degeneration under hypoxia resulted after injury. 3) NF-κB pathway is one of critical pathway of the MMP-2 release in the ACL fibroblasts.In these studies, the production and underlying signaling pathway for MMP-2 were investigated. Further studies showed that the induced MMP-2 was inhibited by Bay11-7082 (NF-ΚB inhibitor), but was not inhibited by PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB203850 (p38 inhibitor).In summary, our results suggested that the differentially effect of microenvironmental factors on ACL might alter the balance in tissue healing, which might in turn be responsible for the ACL poor healing ability. We believe that improving the microenvironment and intervention of MMPs production in the joint fluid after knee injury would be very important in the tissue injury/remodeling processes of human knee.更多还原