【作者】 谢静；

【导师】 宋国立；

【作者基本信息】 重庆大学 ， 生物医学工程， 2012， 博士

【摘要】 前交叉韧带（Anterior cruciate ligament,ACL）和内侧副韧带（Medial collateralligament, MCL）是两个极容易受损伤的膝关节组织。损伤的内侧副韧带具有较强的自我修复能力，即使在韧带中部完全撕裂的情况下，在一个月的时间内也能恢复并保留原有韧带的功能。而损伤的前交叉韧带自我恢复能力极弱，精心的外科手术治疗也不能恢复其功能。前交叉韧带的损伤会引起膝关节的不稳定、膝关节疼痛，甚至还会导致膝关节内其他组织的进一步降解和骨性关节炎（Osteoarthritis,OA）的发生。韧带损伤后的修复过程，都会伴随损伤的细胞外基质的降解和新的细胞外基质的合成。基质金属蛋白酶家族（Matrix Metalloproteinases, MMPs）在这个降解与合成的动态平衡中起着重要的作用。与此同时，越来越多的证据表明赖氨酰氧化酶家族（Lysyl Oxidase Family, LOXs）与组织的损伤、修复有直接的联系。赖氨酰氧化酶催化胶原蛋白和弹性蛋白的赖氨酸肽残基使其变成乙醛肽，从而引起这些不稳定的乙醛肽之间的相互交叉连接，形成不溶的细胞外基质蛋白网。赖氨酰氧化酶的这种能力，使它担当起了细胞外基质生成、稳定和修复的职能。赖氨酰氧化酶家族的每个成员（LOX和LOXL-1,2,3,4）的这种促进胶原之间相互连接的缩合反应功能，到目前为止已经陆续得到证实。但是，赖氨酰氧化酶家族在前交叉韧带和内侧副韧带中的表达情况的研究还不为人们所知。韧带损伤后会出现炎症反应时期（Inflammatory phase）。炎症反应时期伴随各种细胞因子（诸如炎症因子TNF-α、IL-1β和生长因子TGF-β₁等）的产生，这些细胞因子作为炎症反应调节者参与韧带的修复。论文通过体外细胞实验，研究赖氨酰氧化酶家族和基质金属蛋白酶家族的表达。本文的主要研究内容和研究结果如下：首先，用力学拉伸模拟体内韧带细胞的损伤，检测损伤后LOX家族的表达。实验用等双轴拉伸装置，对ACL和MCL细胞同时施加不同程度的力学刺激（6%生理拉伸组；12%损伤拉伸组），结果显示，力学刺激上调LOX家族的表达，且12%的力学损伤使LOX家族的表达远高于6%的生理拉伸。6%生理拉伸时，LOX家族成员在ACL和MCL细胞中都呈上调趋势，但LOX和LOXL-4在ACL中的表达高于MCL，LOXL-3在MCL中的表达高于ACL，而LOXL-1和LOXL-2则有一定的波动性。12%力学损伤时，LOX家族在MCL细胞中的高表达均明显高于在ACL中的表达。其次，在外源性转化生长因子TGF-β₁、肿瘤坏死因子TNF-α、白介素-1beta（IL-1β）作用下，考察正常ACL和MCL细胞中LOX家族的差异性表达。结果显示，TGF-β₁和IL-1β均上调LOX家族的表达，且LOX家族在MCL细胞中的表达总体上高于在ACL中的表达。TGF-β₁引起LOX家族在两种分细胞中的高表达是一个持续的过程；而IL-1β上调LOX家族则呈剂量依赖性。TNF-α抑制LOX家族在两种细胞中的表达，且在ACL细胞中的抑制效果比MCL明显。再次，在损伤的ACL和MCL细胞中（即12%力学拉伸实现细胞损伤），考察TGF-β₁、TNF-α、和IL-1β作用下LOX家族的差异性表达。结果发现，TGF-β₁和IL-1β均增强了LOX家族在损伤的ACL和MCL中的表达，且TGF-β₁的作用效果强于IL-1β。TGF-β₁引起损伤的ACL和MCL细胞中LOXL-1，3和4的高表达强于TGF-β₁对正常细胞的刺激，而LOX和LOXL-2弱于TGF-β₁对正常细胞的刺激。IL-1β上调正常ACL细胞中LOX家族的表达高于损伤的ACL细胞，而损伤的MCL中IL-1β上调LOX家族远高于正常的MCL。TNF-α促使损伤的ACL和MCL中LOX家族（LOXL-2除外）出现峰值后迅速下降，作用24小时后基本低于正常对照组。LOXL-2在损伤的细胞中受TNF-α刺激后一直呈下降趋势，但弱于TNF-α对正常细胞的下调。接着，在实验室已有研究的基础上，在损伤的ACL和MCL细胞中，考察MMP家族部分成员受TGF-β₁、TNF-α、IL-1β作用后的差异性表达。结果显示，三种因子都高表达MMP-1，2，3和12，且MMPs在ACL中的表达均高于在MCL中的表达。炎症因子TNF-α和IL-1β上调了MMPs的基因表达，且TNF-α引起的上调比IL-1β高；而酶谱检测MMP-2蛋白时，发现IL-1β引起的MMP-2活性带的表达比TNF-α强。生长因子TGF-β₁同样引起MMP-1，2，3和12在损伤的ACL和MCL中的高表达，且ACL中的表达均比MCL中高。接着，为了进一步模拟体内环境，实验用滑膜细胞和前交叉韧带细胞实现共培养，考察共培养条件下，损伤的前交叉韧带中MMP-2的蛋白表达。结果显示，共培养促进MMP-2在ACL细胞中的表达，但共培养引起的MMP-2高表达低于单损伤条件引起的MMP-2高表达，而共培养下损伤的ACL中MMP-2的高表达强于共培养下正常ACL的表达或单培养下损伤ACL中的表达。最后，实验采用MMP抑制剂作用于共培养下损伤的前交叉韧带细胞（或损伤的滑膜细胞），结果发现，6种抑制剂均不同程度下调MMP-2在损伤的前交叉韧带细胞或滑膜细胞中的表达，其中以通路NF-kappa B的抑制剂Bay11-7082和AP-1的抑制剂Curcumin效果最为明显，MMP-2的活化形式被抑制，仅存在少量MMP-2的酶原形式的表达。综合以上研究结果，我们证实，与MCL相比，生长因子TGF-β₁和炎症因子TNF-α、IL-1β均引起正常/损伤的ACL细胞表达相对较低的LOX家族，使损伤后的细胞外基质朝着不利于修复的方面发展，我们推断这可能是ACL不能自我修复的原因之一；同时三种因子均引起损伤的ACL中MMPs的高表达远超过损伤的MCL，过高表达的MMPs使损伤后的细胞外基质朝着有利于降解的方面发展，我们推断这可能是ACL不能自我修复的另一个原因。研究LOX家族在损伤ACL和MCL中的表达，可以为临床上韧带创伤的治疗提供定量的参考依据。增强LOX家族在损伤的韧带细胞中的作用，也可能为ACL的修复提供一种方法；同时共培养实现更深层次体内环境的模拟，在共培养下，多种抑制剂均能下调损伤的ACL中MMPs的表达，这可能为ACL治疗提供一种思路。更多还原

【Abstract】 The anterior cruciate ligament （ACL） and medial collateral ligament （MCL） aretwo commonly injured areas of knee joints. In general, the MCL has the capacity toself-heal and restore the joint function of a completely ruptured mid-substance withinone month, and in most cases, without the need for surgery. On the other hand, the ACLdoes not heal satisfactorily, even if the surgical repair is attempted, and ACL injuriesoften lead to knee instability, pain, and even progressive degeneration of other jointtissues and Osteoarthritis （OA）.During lgament healing process, a fine balance between the synthesis ofextracellular matrix （ECM） and its degradation by a large family of enzymes, known asmatrix metalloproteinases （MMPs） of which is well known that the most important roleis that they could cleave one or more of the components of the extracellular matrix ofligaments, is required to maintain the structural integrity of healing tissue; Meanwhile,more and more evidences have inferred the relationship between lysyl oxidase family（LOXs） of which has the capability of oxidizing peptidyl lysine to peptidyl aldehyderesidues within collagen and elastin, thus initiating formation of the covalentcross-linkages that insolubilize these extracellular proteins, and facilitates the formationand repair of extracellular matrix （ECM）, and wound healing. The role of each familymember towards ECM cross-linkage has been consecutively identified. However, thevariations in expression of this LOX family in the normal and injured anterior cruciateligament （ACL） are not fully known.Inflammatory phase accompany with the ligament injury. In the inflammatoryphase, many factors such as transforming growth factor-beta1（TGF-β₁）, tumor necrosisfactor alpha （TNF-α） and interleukin-1beta （IL-1β） involved in mediating the healingprocess. We detected differential expressions of lysyl oxidases and matrixmetalloproteinases in injured ACL-MCL model in vitro. The main research and resultswere as follows:Firstly, we used equi-biaxial stretch system to mimic ligament injury in vivo anddetected the lysyl oxidases expressions in injured ACL and MCL （6%stretchrepresentative of physiological state and12%stretch representative of injurious state）.The results had shown that mechanical stretch up-regulated the expressions of LOXs,moreover,12%stretch-induced up-regulations of LOXs were much higher than6% stretch. In physiological state, LOX and LOXL-4were expressed higher in ACL thanthese in MCL fibroblasts, while LOXL-3in MCL was higher compared with in ACL,the other two members showed fluctuated. In injurious state, LOXs in MCL were moreprominent than those in ACL fibroblasts.Secondly, we detected the differential expressions of lysyl oxidases in normal ACLand MCL fibroblasts induced by TGF-β₁, TNF-α and IL-1β. The results had shown thatboth TGF-β₁and IL-1β could increase the expressions of lysyl oxidases in normal ACLand MCL fibroblasts, and the two factors enhaced LOXs in MCL were much higherthan in ACL. The TGF-β₁-induced-increases of LOXs showed persistent in both ACLand MCL, while IL-1β-induced-increases of LOXs showed a dose-dependent increase.TNF-α down-regulated expressions of lysyl oxidases, furthermore, decreases of LOXsin ACL were more prominent in ACL than in MCL fibroblasts.Thirdly, we detected the differential expressions of lysyl oxidases in ACL andMCL fibroblasts after12%stretch injury induced by TGF-β₁, TNF-α and IL-1β. Wefound that TGF-β₁and IL-1β enhaced different expressions of LOXs in both ACL andMCL after12%stretch injury, moreover, the effects of TGF-β₁on injured fibroblastswere much stronger than IL-1β. LOXL-1,3and4induced by TGF-β₁were much higherin injured fibroblasts than normal cells, LOX and LOXL-2relatively lower in injuredfibroblast than normal ones. IL-1β induced much higher expressions of LOXs in normalACL compared with the injured cells, while in MCL, IL-1β induced much higherexpressions of LOXs in injured MCL than normal cells. TNF-α induced value peaks inLOXs （except LOXL-2） at2hour after treatments, and then immediatelydown-regulated blew non-treated normal controls within24hours. The expression ofTNF-α-induced LOXL-2in injured fibroblasts was constantly down-regulated, but thedown-regulation was somewhat weaker than TNF-α-induced LOXL-2in normal cells.Fourthly, based on the previous data in our lab, we detected the expressions ofMMPs in ACL and MCL after12%stretch injury induced by TGF-β₁, TNF-α and IL-1β.We found these three factors all increased the expressions of MMP-1,2,3, and12inboth ACL and MCL fibroblasts. Inflammatory factors TNF-α and IL-1β bothup-regulated gene expressions of MMPs, moreover, the effect of TNF-α was strongerthan IL-1β. But protein expression of MMP-2by zymography showed the active formof MMP-2was much stronger induced by IL-1β than TNF-α. TGF-β₁also up-regulatedMMP-1,2,3, and12in both ACL and MCL, but TGF-β₁-induced-MMPs in ACL werehigher compared with in MCL. Fifthly, to mimic micro-envirenment, we established coculture system by transwellbwtween ACL fibroblasts and synovial cells. We found that coculture promoted MMP-2expressions. But coculture-induced increase of MMP-2was somewhat lower comparedwith high expression of MMP-2in injured ACL. We combined12%injury stretch andcoculture, found the expression of MMP-2was higher than single coculture or singleinjury.Finally, to resovle the high degradation induced by high expression of MMP-2ininjured ACL, we used signal pathway inhibitors aiming to decrease the high expressionof MMP-2induced by injury. Under coculutre system, we found all6inhibitors in theexperiments could reduce MMP-2expression, especially Bay11-7082, inhibitor ofNF-kappa B, and Curcumin, inhibitor of AP-1, which almostly inhibited the active formof MMP-2, and restore parts of the pro-form.In summary, we comfirmed that TGF-β₁, TNF-α, and IL-1β could inducedrelatively lower expressions of LOXs in normal and injured ACL fibroblasts comparedwith MCL, which mean to count against the repair of injured ligament. We inferred thatrelatively low expressions of LOXs might be on of the reseans why ACL could not selfheal. On the other hand, the three factors respectively promoted high MMPs in injuredACL compared with MCL fibroblasts, which mean to trend to degradation. We inferredthat high expressions of MMPs might be another reason why injured ACL had poorself-healing.The results of LOXs expressions in normal/injured ACL and MCL fibroblastsmight provide the basal data for cure of ligament injury in clinical trial. To enhance theexpressions of LOXs in injured ACL might provide a novel view for ACL healing;meanwhile, to reduce the expressions of MMPs in injured ACL by inhibitors undermimicked micro-envirenment might provide another therapeutial potential of injuredACL ligament.更多还原