膝前交叉韧带重塑部位及其分子机制初探

发布时间：2018-03-25 16:40

本文选题：膝前交叉韧带　切入点：基质金属蛋白酶　出处：《南华大学》2013年硕士论文

【摘要】：目的：本文研究前交叉韧带不同分束在前交叉韧带损伤修复中的作用，探讨是否前交叉韧带的前内侧在前交叉韧带中较后外侧束作用更大。方法：随机抽取本院2010年6月至2012年10月具有术前MRI资料并进行膝关节镜探查的病例进行回顾性分析，通过膝关节MRI矢状位及斜矢状位双维扫描，检测前交叉韧带前内侧束、后外侧束的长度、厚度、宽度及胫骨止点之前和胫骨止点之后的长度。在无菌环境下，从10具自愿捐赠的新鲜尸体上取膝关节，并取出膝关节交叉前韧带，根据ACL的解剖形态学特点和膝关节不同伸屈角度下的紧张状态，将ACL分成前内侧束和后外侧束两个功能纤维束，，束间被疏松结缔组织和血管分离。通过生物动力学测试了前交叉韧带不同部位的结构和力学特点，在肌腱恢复后行拉断试验，记录负荷-延长曲线，并根据此前测得的初长度及横截面积按以下公式计算最大应变和最大应力；提取总RNA，并反转录成cDNA后，应用实时定量PCR检测前内侧束和后外侧束中与肌腱修复相关的常见MMPs基因；再通过将ACL的前内侧束和后外侧束两个功能纤维束，经固定、石蜡包埋，利用MMP-2和MMP-3的多克隆抗体为一抗，进行免疫组化实验；将前内侧束和后外侧束两个功能纤维束中的组织分别收集，匀浆后提取总蛋白，用Western blotting分析不同分束中MMP-2、MMP-3的表达量，将WB条带进行灰度扫描，灰度和条带面积的乘积即为各蛋白相对表达水平。检测了MMPs在前交叉韧带不同部位中的表达水平。结果： 1.通过影像学和生物动力学测试了前交叉韧带不同部位的结构和力学特点；前内束的最大应力和最大应变比后外束的大，进一步说明前交叉韧带的胫骨止点的前部具有成为临床肌腱重建最佳部位的潜力，为今后的临床应用提供理论基础。 2.用RT-PCR、免疫组化、western-blot检测了MMPs在前交叉韧带不同部位中的表达水平，结果表明MMP-2在前内束表达较高，在后外侧束的表达较低；MMP-3在前内侧束表达较低，在后外侧束的表达较高，进一步说明包含胫骨前端的前内侧束比后外侧束更具有重塑潜力。结论：包含胫骨前端的前内侧束比后外侧束更为重要，ACL胫骨止点前方更具有成为临床肌腱修复和重塑的潜在位点。
[Abstract]:Aim: to study the role of different anterior cruciate ligament bundles in the repair of anterior cruciate ligament injury and to explore whether the anterior medial anterior cruciate ligament is more effective than the posterolateral bundle in the anterior cruciate ligament. Methods: from June 2010 to October 2012, patients with preoperative MRI data and knee arthroscopy were randomly selected for retrospective analysis. The anterior medial anterior cruciate ligament (ACL) anterior medial bundle was examined by MRI sagittal and oblique sagittal scan. The length, thickness, width and length of the posterolateral bundle before and after the tibial stop. Under sterile conditions, the knee joint was removed from 10 fresh cadavers donated voluntarily and the cruciate anterior ligament of the knee joint was removed. According to the anatomical morphological characteristics of ACL and the tension of knee joint at different extension and flexion angles, ACL was divided into two functional fiber bundles, the anteromedial bundle and the posterolateral bundle. The structures and mechanical characteristics of different parts of the anterior cruciate ligament (ACL) were measured by biomechanical method. The tensile test was performed after the tendon was restored, and the load-prolongation curves were recorded. The maximum strain and maximum stress were calculated according to the previously measured initial length and cross-sectional area, and the total RNAs were extracted and reversed into cDNA. Real-time quantitative PCR was used to detect the common MMPs genes associated with tendon repair in the anteromedial and posterolateral bundles, and then fixed and paraffin embedded the two functional fiber bundles of ACL, the anterior medial bundle and the posterolateral bundle. Using the polyclonal antibody of MMP-2 and MMP-3 as the first antibody, the immunohistochemical experiment was carried out, the tissues of the anterior medial bundle and the posterolateral bundle were collected separately, the total protein was extracted from homogenate, and the expression of MMP-2mpMMP-3 in different bundles was analyzed by Western blotting. The product of gray scale and band area is the relative expression level of each protein, and the expression level of MMPs in different parts of anterior cruciate ligament is detected. Results:. 1. The structures and mechanical characteristics of different parts of anterior cruciate ligament (ACL) were measured by imaging and biomechanics, the maximum stress and strain of anterior internal bundle were larger than that of posterior external bundle. It is further demonstrated that the anterior tibial insertion of the anterior cruciate ligament has the potential to be the best site for clinical tendon reconstruction, which provides a theoretical basis for clinical application in the future. 2. The expression of MMPs in different parts of anterior cruciate ligament (ACL) was detected by RT-PCR and Western blot. The results showed that the expression of MMP-2 was higher in the anterior medial bundle, lower in the posterolateral bundle, and higher in the anteromedial bundle and posterolateral bundle. It is further demonstrated that the anteromedial bundle containing the anterior tibia has more remodeling potential than the posterolateral bundle. Conclusion:. The anterior medial bundle containing the front of the tibia is more important than the posterolateral bundle. The anterior end of the ACL tibia has a potential site for clinical tendon repair and remodeling.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R686.5

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