本文選題：賴氨酰氧化酶 + 基質(zhì)金屬蛋白酶　； 參考：《重慶大學(xué)》2012年碩士論文

【摘要】：前交叉韌帶（anterior cruciate ligament,ACL）損傷修復(fù)一直是臨床和科學(xué)上的一大難題。由于其自身愈合能力差以及臨床治療的局限性使許多研究者們通過尋找新的途徑來治療修復(fù)破壞的前交叉韌帶。韌帶的損傷修復(fù)過程非常復(fù)雜，包括舊細(xì)胞外基質(zhì)（Extracellular Matrix, ECM）的降解和新細(xì)胞外基質(zhì)的合成，并有多種蛋白酶參與了此過程。如：在細(xì)胞外基質(zhì)起膠原交聯(lián)作用的關(guān)鍵酶—賴氨酰氧化酶(LOXs)和在細(xì)胞外基質(zhì)起降解作用的關(guān)鍵酶—基質(zhì)金屬蛋白酶（MMPs）在此過程中扮演非常重要的角色。因此，研究MMPs和LOXs在正常/損傷前交叉韌帶成纖維細(xì)胞中的的分子響應(yīng)機制可能是探索前交叉韌帶損傷修復(fù)機理的的主要途徑。本課題組在前面的實驗中對正常/損傷ACL成纖維細(xì)胞中的關(guān)鍵酶LOXs和MMPs的基因和蛋白表達已經(jīng)做了一定的研究，但他們所有的研究都是基于在單層培養(yǎng)上的，有很大的局限性。從解剖學(xué)上來看，維持膝關(guān)節(jié)功能的多種組織不是孤立存在的，而是彼此之間相互影響的。ACL是由一層薄的滑膜組織所包裹的，自身的血管床不豐富，需要靠滑膜提供一些營養(yǎng)和血供。當(dāng)滑膜組織損傷后，ACL就暴露在滑液，并且會產(chǎn)生出血性分解產(chǎn)物和蛋白酶，所以為了最大程度地模擬膝關(guān)節(jié)內(nèi)微環(huán)境，我們首先建立ACL成纖維細(xì)胞與滑膜細(xì)胞共培養(yǎng)體系來觀察ACL細(xì)胞與滑膜細(xì)胞之間在共培養(yǎng)條件下是否存在交流？本實驗采用了三種方法，包括：Semi-quantitative PCR、Quantitative real-time PCR和明膠酶譜。本實驗結(jié)果表明： 1）與單層細(xì)胞培養(yǎng)，共培養(yǎng)體系不僅保持了更好的細(xì)胞生長狀態(tài)還能促進細(xì)胞細(xì)胞的遷移。 2）與滑膜共培養(yǎng)下，LOXs和MMP-2在ACL成纖維細(xì)胞中高表達，而MMP-1和MMP-3表達相對降低了。 3）共培養(yǎng)能夠促進損傷ACL成纖維細(xì)胞中的LOXs和MMP-1,2,3表達，尤其是MMP-2的表達。從明膠酶譜可以看出來，，在12%力學(xué)拉伸損傷和共培養(yǎng)共同作用下的MMP-2的蛋白活性表達明顯高于其他組。 這些結(jié)果均表明細(xì)胞與細(xì)胞之間的相互作用在損傷修復(fù)過程中起著非常重要的作用。在單培養(yǎng)和共培養(yǎng)不同培養(yǎng)條件下，LOXs與MMPs的表達出現(xiàn)如此大的差異，說明無論在正常狀態(tài)還是損傷狀態(tài)下，ACL成纖維細(xì)胞和滑膜細(xì)胞之間交流對基因和蛋白的表達起著重要作用。因此，我們根據(jù)間接共培養(yǎng)的特點來推測：在共培養(yǎng)條件下，由于細(xì)胞與細(xì)胞之間的交流，促使細(xì)胞通過旁分泌的形式分泌出的某些細(xì)胞因子能夠誘導(dǎo)另一種細(xì)胞的遷移，并且調(diào)節(jié)細(xì)胞中的LOXs和MMP-1,2,3的表達，這可能會影響ACL組織的損傷后的修復(fù)作用，對前交叉韌帶損傷修復(fù)的機理有著非常重要的臨床意義。
[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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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