共培养下LOXs和MMP-1,2,3在ACL成纤维细胞中的表达

发布时间：2018-05-09 18:37

本文选题：赖氨酰氧化酶 + 基质金属蛋白酶　；参考：《重庆大学》2012年硕士论文

【摘要】：前交叉韧带（anterior cruciate ligament,ACL）损伤修复一直是临床和科学上的一大难题。由于其自身愈合能力差以及临床治疗的局限性使许多研究者们通过寻找新的途径来治疗修复破坏的前交叉韧带。韧带的损伤修复过程非常复杂，包括旧细胞外基质（Extracellular Matrix, ECM）的降解和新细胞外基质的合成，并有多种蛋白酶参与了此过程。如：在细胞外基质起胶原交联作用的关键酶—赖氨酰氧化酶(LOXs)和在细胞外基质起降解作用的关键酶—基质金属蛋白酶（MMPs）在此过程中扮演非常重要的角色。因此，研究MMPs和LOXs在正常/损伤前交叉韧带成纤维细胞中的的分子响应机制可能是探索前交叉韧带损伤修复机理的的主要途径。本课题组在前面的实验中对正常/损伤ACL成纤维细胞中的关键酶LOXs和MMPs的基因和蛋白表达已经做了一定的研究，但他们所有的研究都是基于在单层培养上的，有很大的局限性。从解剖学上来看，维持膝关节功能的多种组织不是孤立存在的，而是彼此之间相互影响的。ACL是由一层薄的滑膜组织所包裹的，自身的血管床不丰富，需要靠滑膜提供一些营养和血供。当滑膜组织损伤后，ACL就暴露在滑液，并且会产生出血性分解产物和蛋白酶，所以为了最大程度地模拟膝关节内微环境，我们首先建立ACL成纤维细胞与滑膜细胞共培养体系来观察ACL细胞与滑膜细胞之间在共培养条件下是否存在交流？本实验采用了三种方法，包括：Semi-quantitative PCR、Quantitative real-time PCR和明胶酶谱。本实验结果表明： 1）与单层细胞培养，共培养体系不仅保持了更好的细胞生长状态还能促进细胞细胞的迁移。 2）与滑膜共培养下，LOXs和MMP-2在ACL成纤维细胞中高表达，而MMP-1和MMP-3表达相对降低了。 3）共培养能够促进损伤ACL成纤维细胞中的LOXs和MMP-1,2,3表达，尤其是MMP-2的表达。从明胶酶谱可以看出来，，在12%力学拉伸损伤和共培养共同作用下的MMP-2的蛋白活性表达明显高于其他组。这些结果均表明细胞与细胞之间的相互作用在损伤修复过程中起着非常重要的作用。在单培养和共培养不同培养条件下，LOXs与MMPs的表达出现如此大的差异，说明无论在正常状态还是损伤状态下，ACL成纤维细胞和滑膜细胞之间交流对基因和蛋白的表达起着重要作用。因此，我们根据间接共培养的特点来推测：在共培养条件下，由于细胞与细胞之间的交流，促使细胞通过旁分泌的形式分泌出的某些细胞因子能够诱导另一种细胞的迁移，并且调节细胞中的LOXs和MMP-1,2,3的表达，这可能会影响ACL组织的损伤后的修复作用，对前交叉韧带损伤修复的机理有着非常重要的临床意义。
[Abstract]:Anterior cruciate ligament (ACL) injury and repair has been a major clinical and scientific problem. Because of its poor healing ability and the limitation of clinical treatment, many researchers seek new ways to repair the damaged anterior cruciate ligament (ACL). The process of ligament repair is very complicated, including the degradation of old extracellular matrix (ECM) and the synthesis of new extracellular matrix. For example, Lysanyl Oxidase (LOXs), the key enzyme of collagen crosslinking in extracellular matrix, and matrix metalloproteinase (MMPs), which play a key role in this process, play a very important role in this process. Therefore, studying the molecular response mechanism of MMPs and LOXs in normal / injured anterior cruciate ligament fibroblasts may be the main way to explore the repair mechanism of anterior cruciate ligament injury. Our team has done some research on the gene and protein expression of the key enzymes LOXs and MMPs in normal / damaged ACL fibroblasts in previous experiments, but all of their studies are based on monolayer culture and have great limitations. Anatomically, the various tissues that maintain the function of the knee are not isolated, but interact with each other. ACL is wrapped in a thin layer of synovial tissue, and its vascular bed is not rich. Synovium is needed to provide some nutrition and blood supply. After synovial tissue injury, ACL is exposed to synovial fluid and produces hemorrhagic decomposition products and proteases, so in order to maximize the simulation of the microenvironment in the knee joint, We first established a co-culture system of ACL fibroblasts and synovial cells to observe whether there was communication between ACL cells and synovial cells under co-culture conditions. Three methods were used in this study, including: Semi-quantitative quantitative real-time PCR and gelatinase spectrum. The results show that: 1) with monolayer cell culture, the co-culture system not only maintained a better cell growth state, but also promoted cell migration. 2) the expression of LOXs and MMP-2 in ACL fibroblasts was higher than that of MMP-1 and MMP-3 under co-culture with synovium. 3) Co-culture could promote the expression of LOXs and MMP-1m2m23 in ACL fibroblasts, especially the expression of MMP-2. It can be seen from the gelatinase spectrum that the protein activity expression of MMP-2 under 12% mechanical tensile injury and co-culture was significantly higher than that of other groups. These results suggest that cell-cell interaction plays a very important role in the process of injury-repair. The difference between the expression of LOXs and MMPs in monoculture and co-culture suggests that the communication between fibroblasts and synovial cells plays an important role in the expression of genes and proteins. Therefore, based on the characteristics of indirect co-culture, we speculate that under co-culture conditions, some cytokines secreted by cells through paracrine can induce the migration of another kind of cells because of the communication between cells and cells. The regulation of the expression of LOXs and MMP-1 / MMP _ 2 ~ (2 +) may affect the repair of ACL tissue after injury, which is of great clinical significance to the repair mechanism of anterior cruciate ligament (ACL) injury.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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